Compositions and methods for inhibition of vegf

ABSTRACT

Disclosed herein are siRNA compositions and methods useful for inhibiting expression of vascular endothelial growth factor (VEGF) isoforms. Diseases which involve angiogenesis stimulated by overexpression of VEGF, such as diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering small interfering RNAs as disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. nonprovisional patentapplication Ser. No. 12/960,933 filed on Dec. 6, 2010 entitled“COMPOSITIONS AND METHODS FOR INHIBITION OF VEGF,” which claims benefitof priority to U.S. Provisional Patent Application No. 61/266,645 filedon Dec. 4, 2009 entitled “COMPOSITIONS AND METHODS FOR INHIBITION OFVEGF,” the entire contents of which are hereby incorporated byreference.

GOVERNMENT INTERESTS

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PARTIES TO A JOINT RESEARCH AGREEMENT

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E. INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

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BACKGROUND

1. Field of Invention

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2. Description of Related Art

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BRIEF SUMMARY OF THE INVENTION

Angiogenesis, defined as the growth of new capillary blood vessels or“neovascularization,” plays a fundamental role in growth anddevelopment. In mature humans, the ability to initiate angiogenesis ispresent in all tissues, but is held under strict control. A keyregulator of angiogenesis is vascular endothelial growth factor(“VEGF”), also called vascular permeability factor (“VPF”). Angiogenesisis initiated when secreted VEGF binds to the Flt-1 and Flk-1/KDRreceptors (also called VEGF receptor 1 and VEGF receptor 2), which areexpressed on the surface of endothelial cells. Flt-1 and Flk-1/KDR aretransmembrane protein tyrosine kinases, and binding of VEGF initiates acell signal cascade resulting in the ultimate neovascularization in thesurrounding tissue.

There are three main different VEGF alternative splice forms (i.e.,isoforms) in humans (VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉), while a number ofother variants also exist (VEGF₂₀₆, VEGF₁₈₃, VEGF₁₄₈, VEGF_(165b) andVEGF₁₄₅) Remarkably, not all of the isoforms are pro-angiogenic. It hasbeen demonstrated that at least VEGF_(165b) is capable of counteractingthe effects of VEGF₁₆₅ induced angiogenesis. Without being bound bytheory, it appears that VEGF_(165b) is capable of preventing VEGFReceptor 2 signaling. As such, secretion of VEGF_(165b) may be able toprevent or retard angiogenesis in pathological states.

Aberrant angiogenesis, or the pathogenic growth of new blood vessels, isimplicated in a number of conditions. Among these conditions arediabetic retinopathy, psoriasis, exudative or “wet” age-related maculardegeneration (“ARMD”), rheumatoid arthritis and other inflammatorydiseases, and most cancers. The diseased tissues or tumors associatedwith these conditions express abnormally high levels of VEGF, and show ahigh degree of vascularization or vascular permeability.

ARMD in particular is a clinically important angiogenic disease. Thiscondition is characterized by choroidal neovascularization in one orboth eyes in aging individuals, and is the major cause of blindness inindustrialized countries.

RNA interference (hereinafter “RNAi”) is a method ofpost-transcriptional gene regulation that is conserved throughout manyeukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide)double stranded RNA (“dsRNA”) molecules which are present in the cell.These short dsRNA molecules, called “short interfering RNA” or “siRNA,”cause the destruction of messenger RNAs (“mRNAs”) which share sequencehomology with the siRNA to within one nucleotide resolution. It isbelieved that the siRNA and the targeted mRNA bind to an “RNA-inducedsilencing complex” or “RISC”, which cleaves the targeted mRNA. The siRNAis apparently recycled much like a multiple-turnover enzyme, with 1siRNA molecule capable of inducing cleavage of approximately 1000 mRNAmolecules. siRNA-mediated RNAi degradation of an mRNA is therefore moreeffective than currently available technologies for inhibitingexpression of a target gene. However, such methods are not directly ableto be translated into therapeutic agents for treatment of disease.

What is needed, therefore, are agents which selectively inhibitexpression of pro-angiogenic VEGF in catalytic or sub-stoichiometricamounts in mammals, while inducing or maintaining the secretion ofanti-angiogenic VEGF isoforms.

The present disclosure is directed to siRNAs which specifically targetand cause RNAi-induced degradation of mRNA from VEGF and its isoforms.The siRNA compounds and compositions of the disclosure are used toinhibit angiogenesis, in particular for the treatment of canceroustumors, age-related macular degeneration, and other angiogenic diseases.

Thus, the disclosure provides an isolated siRNA which targets human VEGFmRNA, or an alternative splice form, mutant or cognate thereof. Forexample, in one embodiment, the siRNA targets pro-angiogenic VEGF mRNAisoforms such as VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, VEGF₂₀₆, VEGF₁₈₃, VEGF₁₄₈,and/or VEGF₁₄₅. In certain embodiments, the siRNA comprises a sense RNAstrand and an antisense RNA strand which form an RNA duplex. The senseRNA strand comprises a nucleotide sequence identical to a targetsequence of about 19 to about 25 contiguous nucleotides in the targetmRNA.

The disclosure also provides recombinant plasmids and viral vectorswhich express the siRNA disclosed herein, as well as pharmaceuticalcompositions comprising such an siRNA and a pharmaceutically acceptablecarrier.

The disclosure further provides a method of inhibiting expression ofhuman pro-angiogenic VEGF mRNA, or an alternative splice form, mutant orcognate thereof, while sparing anti-angiogenic VEGF mRNA, comprisingadministering to a subject an effective amount of siRNA such that thetarget mRNA is degraded.

The disclosure further provides a method of inhibiting angiogenesis in asubject, comprising administering to a subject an effective amount of ansiRNA targeted to pro-angiogenic human VEGF mRNA or an alternativesplice form, mutant or cognate thereof.

The disclosure further provides a method of treating an angiogenicdisease, comprising administering to a subject in need of such treatmentan effective amount of an siRNA targeted to human proangiogenic VEGFmRNA or an alternative splice form, mutant or cognate thereof, such thatangiogenesis associated with the angiogenic disease is inhibited.

DESCRIPTION OF DRAWINGS

The file of this patent contains at least one photograph or drawingexecuted in color. Copies of this patent with color drawing(s) orphotograph(s) will be provided by the Patent and Trademark Office uponrequest and payment of necessary fee.

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings, in which:

FIGS. 1A and 1B are a histograms of VEGF concentration (in pg/ml) inhypoxic 293 and HeLa cells treated with no siRNA (“-”); nonspecificsiRNA (“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). VEGFconcentration (in pg/ml) in non-hypoxic 293 and HeLa cells is alsoshown. Each bar represents the average of four experiments, and theerror is the standard deviation of the mean.

FIG. 2 is a histogram of murine VEGF concentration (in pg/ml) in hypoxicNIH 3T3 cells treated with no siRNA (“-”); nonspecific siRNA(“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). Each barrepresents the average of six experiments and the error is the standarddeviation of the mean.

FIG. 3 is a histogram of human VEGF concentration (pg/total protein) inretinas from mice injected with adenovirus expressing human VEGF(“AdVEGF”) in the presence of either GFP siRNA (dark gray bar) or humanVEGF siRNA (light grey bar). Each bar represents the average of 5 eyesand the error bars represent the standard error of the mean.

FIG. 4 is a histogram showing the mean area (in mm²) of laser-inducedCNV in control eyes given subretinal injections of GFP siRNA (N=9; “GFPsiRNA”), and in eyes given subretinal injections of mouse VEGF siRNA(N=7; “Mouse VEGF siRNA”). The error bars represent the standard errorof the mean.

FIG. 5 is a schematic representation of pAAVsiRNA, a cis-acting plasmidused to generate a recombinant AAV viral vector of the invention. “ITR”:AAV inverted terminal repeats; “U6”: U6 RNA promoters; “Sense”: siRNAsense coding sequence; “Anti”: siRNA antisense coding sequence; “PolyT”:polythymidine termination signals.

FIG. 6 shows histograms of the mean area (in mm²) of laser-induced CNVin treatment in mouse eyes injected (A) subretinally or (B)intravitreally with a mouse anti-VEGF siRNA (“mVEGF1.siRNA”) or controlsiRNA (“GFP1.siRNA”). The error bars represent the standard error of themean. (C) is a histogram of the mean area (in mm²) of laser-induced CNVin mouse eyes injected intravitreally with: phosphate-buffered salinewith no siRNA at 1 day post-laser induction (“PBS”; CNV area measured at14 days post-laser induction); control siRNA at 14 days post-laserinduction (“GFP1.siRNA”; CNV area measured at 21 days post-laserinduction); or a mouse anti-VEGF siRNA at 14 days post-laser induction(“mVEGF1.siRNA”; CNV area measured at 21 days post-laser induction). Theerror bars represent the standard error of the mean.

FIG. 7 is a graph of the percent of VEGF (“% VEGF”) protein in mouseeyes injected sub-retinally with human anti-VEGF siRNA (“Cand5”) andcontrol siRNA (“GFP1.siRNA”) at 0 (n=2; pre-siRNA injection), 6 (n=3),10 (n=3) and 14 (n=3) days post-injection. % VEGF=([VEGF] in the Cand5eye/[VEGF] in the GFP1.siRNA eye)*100.

FIG. 8 is a graph of hVEGF protein levels in 293 cells transfected withtransfected with human VEGF siRNAs, non-specific siRNA (EGFP siRNA) ormock transfections without siRNA.

FIG. 9 is a graph of the dose response studies with Cand5 (bevasrianib),hVEGF#1, hVEGF#2, hVEGF#3, hVEGF#4, hVEGF#6 and hVEGF#7.

FIG. 10 is a schematic of the various isoforms of VEGF and their exonusage.

FIG. 11 is a diagram comparing the homology of VEGF₁₆₅ and VEGF_(165b)at the exon 7/8 junction.

FIG. 12 depicts the amount of VEGF protein expressed for various siRNAstargeting the VEGF₁₆₅ exon 7/8 junction.

FIG. 13 depicts the percent knockdown of human VEGF protein for varioussiRNAs targeting the VEGF₁₆₅ exon 7/8 junction.

FIG. 14 depicts the amount of VEGF protein expressed for a secondaryscreen of siRNAs targeting the VEGF₁₆₅ exon 7/8 junction.

FIG. 15 depicts the percent knockdown of human VEGF protein for asecondary screen of siRNAs targeting the VEGF₁₆₅ exon 7/8 junction.

FIG. 16 depicts the percent knockdown of human VEGF protein for asecondary screen of siRNAs targeting the VEGF₁₆₅ exon 7/8 junction atvarying concentrations.

FIG. 17 depicts the percent knockdown of human VEGF protein over sevendays for a secondary screen of siRNAs targeting the VEGF₁₆₅ exon 7/8junction.

FIG. 18 depicts the effect of siRNA targeting the VEGF₁₆₅ exon 7/8junction on GAPDH mRNA expression using RT-PCR.

FIG. 19 depicts the effect of siRNA targeting the VEGF₁₆₅ exon 7/8junction on VEGF₁₆₅ mRNA expression using RT-PCR.

FIG. 20 depicts the effect of siRNA targeting the VEGF exon 7/8 junctionon VEGF₁₈₉ mRNA expression using RT-PCR.

FIG. 21 depicts the effect of siRNA targeting the VEGF exon 7/8 junctionon VEGF₁₂₁ mRNA expression using RT-PCR.

FIG. 22 depicts the effect of siRNA targeting the VEGF₁₆₅ exon 7/8junction on VEGF_(165b) mRNA expression using RT-PCR. Double banding atabout 600 bp is artefactual.

FIG. 23 depicts the cytokine profile of ARPE19 cells following treatmentwith selected siRNAs.

FIG. 24 depicts the effect of siRNAs on total VEGF protein secretion byARPE19 cells.

FIG. 25 depicts the effect of siRNAs on total VEGF protein secretion byARPE19 cells.

FIG. 26 depicts the effect of siRNAs on total VEGF protein secretion byARPE19 cells.

FIG. 27 depicts the effect of siRNAs on total VEGF protein secretion byARPE19 cells.

FIG. 28 depicts the stability of bevasiranib under different temperatureconditions over time.

FIG. 29 depicts the stability of bevasiranib under different temperatureconditions over time.

FIG. 30 depicts the stability of OPK-HVB-004 under different temperatureconditions over time.

FIG. 31 depicts the stability of OPK-HVB-009 under different temperatureconditions over time.

FIG. 32 depicts the stability of OPK-HVB-010 under different temperatureconditions over time

FIG. 33 depicts the homology between human, rat and mouse VEGF sequencesat the 3′ terminal end.

FIG. 34 depicts the effect of siRNAs on rat VEGF secretion by C6 cells.

FIG. 35 depicts the effect of siRNAs on mouse VEGF secretion by NIH3T3cells.

FIG. 36 depicts the effect of siRNAs on VEGF secretion by ARPE19 cells.

FIG. 37 depicts the effect of siRNAs on VEGF secretion by ARPE19 cells.

FIG. 38 depicts the effect of siRNAs on VEGF secretion by ARPE19 cells.

FIG. 39 depicts the effect of siRNAs on mouse VEGF secretion by NIH3T3cells

FIG. 40 depicts the effect of siRNAs on VEGF mRNA message in ARPE19cells

FIG. 41 depicts the effect of siRNAs on VEGF expression in C6 cells.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “molecule” is a reference to one or more molecules and equivalentsthereof known to those skilled in the art, and so forth. As used herein,the term “about” means plus or minus 10% of the numerical value of thenumber with which it is being used. Therefore, about 50% means in therange of 45%-55%.

As used herein, a “subject” includes a human being or non-human animal.In certain embodiments, the subject is a human being.

As used herein, an “effective amount” of the siRNA is an amountsufficient to cause RNAi-mediated degradation of the target mRNA incell. The term clinically effective amount is an amount that whenadministered to a subject, will inhibit the progression of angiogenesisin a subject by RNA silencing.

As used herein, “isolated” means altered or removed from the naturalstate through human intervention. For example, an siRNA naturallypresent in a living animal is not “isolated,” but a synthetic siRNA, oran siRNA partially or completely separated from the coexisting materialsof its natural state is “isolated.” An isolated siRNA can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a cell into which the siRNA has been delivered.

As used herein, “target mRNA” means an mRNA comprising a complementarysense sequence to an siRNA antisense strand. Such a target mRNA need notbe 100% homologous to the siRNA antisense strand, as long as the siRNAfunctions to silence or otherwise form a RISC complex with the targetmRNA. Target mRNAs of particular use in the methods of the disclosureinclude, for example, pro-angiogenic VEGF mRNA isoforms such as VEGF₁₂₁,VEGF₁₆₅, and VEGF₁₈₉, VEGF₂₀₆, VEGF₁₈₃, VEGF₁₄₈, and VEGF₁₄₅ andcombinations thereof. In certain other embodiments, the target mRNA doesnot comprise anti-angiogenic VEGF_(165b) mRNA, but targets at least oneother VEGF isoforms.

As used herein the term “partially non-complementary” is intended tomean an siRNA sequence which although, perhaps sharing some sequencehomology to a non-target sequence still differs sufficiently such thatRNA silencing does not occur for the non-target sequence. Partiallynon-complementary include sequences that are 90% homologous, 85%,homologous, 80% homologous, 75% homologous, 70% homologous, 65%homologous, 60%, homologous, 55% homologous, 50% homologous, 45%homologous, 40% homologous, 35%, homologous, 30% homologous, 25%homologous, 20% homologous, 15% homologous, 10%, homologous, 5%homologous, 2% homologous, and 1% homologous to a non-target sequence. Asequence that is entirely non-homologous to a non-target sequence isconsidered non-complementary to the sequence.

As used herein, a gene or mRNA which is “cognate” to human VEGF or mRNAfrom another mammalian species which is homologous to human VEGF. Forexample, the cognate VEGF mRNA from the mouse is given in SEQ ID NO: 1.

Unless otherwise indicated, all nucleic acid sequences herein are givenin the 5′ to 3′ direction. Also, all deoxyribonucleotides in a nucleicacid sequence are represented by capital letters (e.g., deoxythymidineis “T”), and ribonucleotides in a nucleic acid sequence are representedby lower case letters (e.g., uridine is “u”).

Compositions and methods comprising siRNA targeted to VEGF and itsvarious isoforms can be used to inhibit angiogenesis, in particular forthe treatment of angiogenic disease. The siRNA are believed to cause theRNAi-mediated degradation of these mRNAs, so that the protein product ofthe VEGF and its isoforms are not produced or is produced in reducedamounts. Because VEGF binding to the Flt-1 or Flk-1/KDR receptors isrequired for initiating and maintaining angiogenesis, the siRNA-mediateddegradation of VEGF and its isoforms as well as Flt-1 or Flk-1/KDR mRNAmay also be used to inhibit the angiogenic process.

One aspect of the present disclosure therefore provides isolated siRNAcomprising short double-stranded RNA from about 17 nucleotides to about29 nucleotides in length, and in certain embodiments from about 19 toabout 25 nucleotides in length, that are targeted to the target mRNA.The siRNA comprise a sense RNA strand and a complementary antisense RNAstrand annealed together by standard Watson-Crick base-pairinginteractions (hereinafter “base-paired”). As is described in more detailbelow, the sense strand comprises a nucleic acid sequence which isidentical or closely homologous to a target sequence contained withinthe target mRNA.

The sense and antisense strands of the siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. Without wishingto be bound by any theory, it is believed that the hairpin area of thelatter type of siRNA molecule is cleaved intracellularly by the “Dicer”protein (or its equivalent) to form an siRNA of two individualbase-paired RNA molecules.

Splice variants of human VEGF are known, including pro-angiogenic VEGFmRNA isoforms such as VEGF₁₂₁(SEQ ID NO: 2), VEGF₁₆₅ (SEQ ID NO: 3), andVEGF₁₈₉(SEQ ID NO: 4), VEGF₂₀₆(SEQ ID NO: 5; GenBank Accession No.C5245579), VEGF₁₈₃ (GenBank Accession No. AJ010438), VEGF₁₄₈ (GenBankAccession No. AF091352), and VEGF₁₄₅ (GenBank Accession No. C5245578),as well as anti-angiogenic VEGF_(165b) mRNA (GenBank Accession No.AF430806). The mRNA transcribed from the human VEGF and its isoforms, aswell as Flt-1 (SEQ ID NO: 6) or Flk-1/KDR (SEQ ID NO: 7) genes can beanalyzed for further alternative splice forms using techniqueswell-known in the art. Such techniques include reversetranscription-polymerase chain reaction (RT-PCR), northern blotting andin-situ hybridization. Techniques for analyzing mRNA sequences aredescribed, for example, in Busting SA (2000), J. Mol. Endocrinol. 25:169-193, the entire disclosure of which is herein incorporated byreference. Representative techniques for identifying alternativelyspliced mRNAs are also described below.

For example, databases that contain nucleotide sequences related to agiven disease gene can be used to identify alternatively spliced mRNA.Such databases include GenBank, Embase, and the Cancer Genome AnatomyProject (CGAP) database. The CGAP database, for example, containsexpressed sequence tags (ESTs) from various types of human cancers. AnmRNA or gene sequence from the VEGF and its isoforms as well as Flt-1 orFlk-1/KDR genes can be used to query such a database to determinewhether ESTs representing alternatively spliced mRNAs have been foundfor a these genes.

A technique called “RNAse protection” can also be used to identifyalternatively spliced VEGF and its isoforms as well as Flt-1 orFlk-1/KDR mRNAs. RNAse protection involves translation of a genesequence into synthetic RNA, which is hybridized to RNA derived fromother cells; for example, cells from tissue at or near the site ofneovascularization. The hybridized RNA is then incubated with enzymesthat recognize RNA:RNA hybrid mismatches. Smaller than expectedfragments indicate the presence of alternatively spliced mRNAs. Theputative alternatively spliced mRNAs can be cloned and sequenced bymethods well known to those skilled in the art.

RT-PCR can also be used to identify alternatively spliced VEGF and itsisoforms as well as Ht-1 or Hk-1/KDR mRNAs. In RT-PCR, mRNA from tissueor cells is converted into cDNA by the enzyme reverse transcriptase,using methods well-known to those of ordinary skill in the art. Thecoding sequence of the cDNA is then amplified via PCR using a forwardprimer located in the 5′ translated region, and a reverse primer locatedin the 3′ translated region. In some embodiments, all the bases encodingthe cDNA are amplified. The amplified products can be analyzed foralternative splice forms, for example by comparing the size of theamplified products with the size of the expected product from normallyspliced mRNA, e.g., by agarose gel electrophoresis. Any change in thesize of the amplified product can indicate alternative splicing.

mRNA produced from mutant VEGF and its isoforms as well as Flt-1 orHk-1/KDR genes can also be readily identified through the techniquesdescribed above for identifying alternative splice forms. As usedherein, “mutant” VEGF and its isoforms as well as Flt-1 or Flk-1/KDRgenes or mRNA include human VEGF and its isoforms as well as Flt-1 orFlk-1/KDR genes or mRNA which differ in sequence from the VEGF and itsisoforms as well as Flt-1 or Flk-1/KDR sequences set forth herein. Thus,allelic forms of these genes, and the mRNA produced from them, areconsidered “mutants” for purposes of this invention.

It is understood that human VEGF and its isoforms, as well as Flt-1 orFlk-1/KDR mRNA may contain target sequences in common with theirrespective alternative splice forms, cognates or mutants. A single siRNAcomprising such a common targeting sequence can therefore induceRNAi-mediated degradation of different RNA types which contain thecommon targeting sequence. For example, as shown in FIG. 10, all VEGFisoforms share exons 1-5. However, in VEGF₁₂₁(SEQ ID NO: 2) exons 6 and7 (7a and 7b) are deleted. In VEGF₁₆₅ (SEQ ID NO: 3) exon 6 (6a and 6b)is deleted. In VEGF₁₈₉ (SEQ ID NO: 4) exon 6b is deleted. In VEGF₁₈₃ aportion of exon 6a is deleted as well as the complete exon 6b sequence.VEGF₁₄₈ has a deletion of exon 6 (6a and 6b) as well as exon 7b and aportion of exon 8. In VEGF₁₄₅ exon 6b and exon 7 (7a and 7b) aredeleted. The only known anti-angiogenic isoform of VEGF, VEGF_(165b),lacks exon 6 (6a and 6b), but additionally comprises a pseudo-exon 9.The pseudo-exon 9 is a result of a reading frame shift caused by thedeletion of a stop codon, thus allowing a portion of the 3′UTR to betranslated as protein. See for example, Bates et al., Can. Res. 62:4123(2002), herein incorporated by reference in its entirety. VEGF₂₀₆ (SEQID NO: 5) is the full length sequence VEGF with no deletions. Thus, incertain embodiments, the siRNA targets one or more isoforms, such asVEGF₁₂₁(SEQ ID NO: 2), VEGF₁₆₅ (SEQ ID NO: 3), and VEGF₁₈₉(SEQ ID NO:4), VEGF₂₀₆(SEQ ID NO: 5; GenBank Accession No. C5245579), VEGF₁₈₃(GenBank Accession No. AJ010438), VEGF₁₄₈ (GenBank Accession No.AF091352), and/or VEGF₁₄₅ (GenBank Accession No. C5245578), but sparesothers, such as VEGF_(165b), because the siRNA targets a shared exonamong certain isoforms but not others.

In one embodiment, provided is an isolated siRNA comprising of a duplexof a first RNA strand and a second RNA strand, said first RNA strandcomprising a nucleotide sequence identical to a target sequence of about19 to about 25 contiguous nucleotides to a vascular endothelial growthfactor (VEGF) isoform selected from the group consisting of humanVEGF₁₂₁, VEGF₁₆₅ VEGF₁₈₉, VEGF₂₀₆, VEGF₁₈₃, VEGF₁₄₈, VEGF₁₄₅ andcombinations thereof; further wherein said siRNA is at least partiallynon-complementary to VEGF_(165b), with the proviso that the human VEGFmRNA is not SEQ ID NO. 42. Further embodiments include methods of usingsuch siRNA to inhibit angiogenesis and pharmaceutical compositionscomprising a therapeutically effective amount of such siRNA to inhibitangiogenesis.

The siRNA can comprise partially purified RNA, substantially pure RNA,synthetic RNA, or recombinantly produced RNA, as well as altered RNAthat differs from naturally-occurring RNA by the addition, deletion,substitution and/or alteration of one or more nucleotides. Suchalterations can include addition of non-nucleotide material, such as tothe end(s) of the siRNA or to one or more internal nucleotides of thesiRNA, including modifications that make the siRNA resistant to nucleasedigestion.

One or both strands of the siRNA can also comprise a 3′ overhang. Asused herein, a “3′ overhang” refers to at least one unpaired nucleotideextending from the 3′-end of a duplexed RNA strand. In some embodiments,the siRNA does not comprise a overhang and has a blunt end. In someembodiments, both ends of the siRNA comprise a blunt end. In someembodiments, the siRNA comprises a 17mer that contiguous with a targetmRNA and dTdT overhang. In some embodiments, the siRNA is a siRNA thatcan inhibit the secretion or production of VEGF from cells fromdifferent species. For example, in some embodiments, the siRNA caninhibit VEGF secretion or inhibition from a human cell, a rat cell,and/or a mouse cell. In some embodiments, the siRNA can inhibit thesecretion or production of VEGF from a mouse cell and a human cell, butnot from a rat cell. In some embodiments, the siRNA can inhibit thesecretion or production of VEGF from a rat cell and a human cell, butnot from a mouse cell. In some embodiments, the siRNA can inhibit thesecretion or production of VEGF from a human cell, a mouse cell, and arat cell. The selectivity of the siRNA can be based upon the homologybetween the different sequences. For example, FIG. 33 shows the homologybetween the terminal codons encoding human, mouse and rat VEGF. Thesedifferences can be exploited to produce siRNAs that can selectivelyinhibit the production of VEGF from one or more species.

In some embodiments, siRNAs comprising less than 21 nucleotides, e.g.17, 18, 19, or 20, can be used to avoid any potential non-specific invivo responses. (See, Ambati, Nature, 452, 591-597 (3 Apr. 2008)). Forexample, siRNAs comprising less than 21 nucleotides can be used to avoidactivating a TLR3 response in vivo.

Thus in one embodiment, the siRNA comprises at least one 3′ overhang offrom 1 to about 6 nucleotides (which includes ribonucleotides ordeoxynucleotides) in length, preferably from 1 to about 5 nucleotides inlength, more preferably from 1 to about 4 nucleotides in length, andparticularly preferably from about 2 to about 4 nucleotides in length.

In the embodiment in which both strands of the siRNA molecule comprise a3′ overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′ overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA can comprise 3′ overhangs ofdithymidylic acid (“TT”) or diuridylic acid

(“uu”).

In order to enhance the stability of the present siRNA, the 3′ overhangscan be also stabilized against degradation. In one embodiment, theoverhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′ hydroxyl in the 2′-deoxythymidinesignificantly enhances the nuclease resistance of the 3′ overhang intissue culture medium.

In certain embodiments, the siRNA comprises the sequence AA(N19)TT orNA(N21), where N is any nucleotide. These siRNA comprise approximately30-70% GC, and preferably comprise approximately 50% G/C. The sequenceof the sense siRNA strand corresponds to (N19)TT or N21 (i.e., positions3 to 23), respectively. In the latter case, the 3′ end of the sensesiRNA is converted to TT. The rationale for this sequence conversion isto generate a symmetric duplex with respect to the sequence compositionof the sense and antisense strand 3′ overhangs. The antisense RNA strandis then synthesized as the complement to positions 1 to 21 of the sensestrand.

Because position 1 of the 23-nt sense strand in these embodiments is notrecognized in a sequence-specific manner by the antisense strand, the3′-most nucleotide residue of the antisense strand can be chosendeliberately. However, the penultimate nucleotide of the antisensestrand (complementary to position 2 of the 23-nt sense strand in eitherembodiment) is generally complementary to the targeted sequence.

In another embodiment, the siRNA comprises the sequence NAR(N17)YNN,where R is a purine (e.g., A or G) and Y is a pyrimidine (e.g., C orU/T). The respective 21-nt sense and antisense RNA strands of thisembodiment therefore generally begin with a purine nucleotide. SuchsiRNA can be expressed from pol III expression vectors without a changein targeting site, as expression of RNAs from pol III promoters is onlybelieved to be efficient when the first transcribed nucleotide is apurine.

In a further embodiment, the siRNA comprises a sequence having no morethan five (5) consecutive purines or pyrimidines. In a furtherembodiment, the siRNA comprises a sequence having no more than five (5)consecutive nucleotides having the same nucleobase (i.e., A, C, G, orU/T).

The siRNA can be targeted to any stretch of approximately 19-25contiguous nucleotides in any of the target mRNA sequences (the “targetsequence”). Techniques for selecting target sequences for siRNA aregiven, for example, in Tuschl T et al., “The siRNA User Guide,” revisedOct. 11, 2002, the entire disclosure of which is herein incorporated byreference. “The siRNA User Guide” is available on the world wide web ata website maintained by Dr. Thomas Tuschl, Department of CellularBiochemistry, AG 105, Max-Planck-Institute for Biophysical Chemistry,37077 Gottingen, Germany, and can be found by accessing the website ofthe Max Planck Institute and searching with the keyword “siRNA.” Thus,the sense strand of the present siRNA comprises a nucleotide sequenceidentical to any contiguous stretch of about 19 to about 25 nucleotidesin the target mRNA.

In some embodiments, the siRNA is 19 nucleotides and comprises 17nucleotides that are identical to a target mRNA. In some embodiments,the siRNA is 19 nucleotides in length comprising at least one blunt end.In some embodiments, each end of the 19mer has a blunt end. In someembodiments, the 19mer comprises at least one dT overhang. In someembodiments, the 19mer comprises two dT overhangs.

Generally, a target sequence on the target mRNA can be selected from agiven cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′ direction) from thestart codon. The target sequence can, however, be located in the 5′ or3′ untranslated regions, or in the region nearby the start codon (see,e.g., the target sequences of SEQ ID NOS: 73 and 74 in Table 1 below,which are within 100 nt of the 5′-end of the VEGF₁₂₁ cDNA.

In a further embodiment of the present invention, the target mRNAsequence comprises no more than five (5) consecutive purines orpyrimidines. For example, a suitable target sequence in the VEGF₁₂₁ cDNAsequence is:

(SEQ ID NO: 8) TCATCACGAAGTGGTGAAG

Thus, an siRNA targeting this sequence, and which has 3′ uu overhangs oneach strand (overhangs shown in bold), is:

(SEQ ID NO: 9) 5′-ucaucacgaaguggugaaguu-3′ (SEQ ID NO: 10)3′-uuaguagugcuucaccacuuc-5′

An siRNA targeting this same sequence, but having 3′ TT overhangs oneach strand (overhangs shown in bold) is:

(SEQ ID NO: 11) 5′-ucaucacgaaguggugaagTT-3′ (SEQ ID NO: 12)3′-TTaguagugcuucaccacuuc-5′

Other VEGF₁₂₁ target sequences from which siRNA can be derived are givenin Table 1. It is understood that all VEGF₁₂₁ target sequences listed inTable 1 are within that portion of the VEGF₁₂₁ alternative splice formwhich is common to all human VEGF alternative splice forms. Thus, theVEGF₁₂₁ target sequences in Table 1 can also target VEGF₁₆₅, VEGF₁₈₉ andVEGF₂₀₆ mRNA. Target sequences which target a specific VEGF isoform canalso be readily identified. For example, a target sequence which targetsVEGF₁₆₅ mRNA but not VEGF₁₂₁ mRNA is AACGTACTTGCAGATGTGACA (SEQ ID NO:13). Conversely, target sequences which target pro-angiogenic VEGF mRNAisoforms such as VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉, VEGF₂₀₆, VEGF₁₈₃,VEGF₁₄₈, and VEGF₁₄₅ and combinations thereof, but does not targetanti-angiogenic VEGF_(165b) mRNA include the sequences found in Table 2,with the proviso that the VEGF mRNA is not SEQ ID No. 42. In certainembodiments, said human VEGF mRNA is selected from the group consistingof SEQ ID NO: 86; SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ IDNO: 90; SEQ ID NO: 91; SEQ ID NO: 92; SEQ ID NO: 93; SEQ ID NO: 94; SEQID NO: 95; SEQ ID NO: 96; SEQ ID NO: 97; and SEQ ID NO: 98. In certainembodiments, said human VEGF mRNA is selected from SEQ ID NO. 88 and SEQID NO. 94.

By selectively targeting the angiogenic isoforms of VEGF, while sparingthe anti-angiogenic isoform, it is possible to enhance theanti-angiogenic effects of siRNA treatment. As shown in FIG. 11, theregion between exon 7 and 9 differ between the angiogenic andantiangiogenic sequences. According to the various embodiments, it ispossible to selectively target this region where the siRNA is at leastpartially complementary to the angiogenic isoforms, but at leastpartially or fully non-complementary to the anti-angiogenic isoform.Consequently, in certain embodiments, the siRNA would not inhibit theexpression of the anti-angiogenic isoform, VEGF_(165b) with the provisothat the VEGF mRNA is not SEQ ID No. 42. In certain embodiments, saidhuman VEGF mRNA is selected from the group consisting of SEQ ID NO: 86;SEQ ID NO: 87; SEQ ID NO: 88; SEQ ID NO: 89; SEQ ID NO: 90; SEQ ID NO:91; SEQ ID NO: 92; SEQ ID NO: 93; SEQ ID NO: 94; SEQ ID NO: 95; SEQ IDNO: 96; SEQ ID NO: 97; SEQ ID NO: 98, SEQ ID NO 99, SEQ ID NO 100, SEQID NO 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO:105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118.In certain embodiments, said human VEGF mRNA is selected from SEQ ID NO.88 and SEQ ID NO. 94.

Exemplary target sequences for human Flt-1 for human Flk-1/KDR are givenin PCT/US2003/0022444 filed Jul. 18, 2003, herein incorporated byreference in its entirety.

TABLE 1 VEGF Target Sequences SEQ SEQ ID ID target sequence NO:target sequence NO: cognate VEGF mRNA sequence 1 GATAGAGCAAGACAAGAAA 26Splice variant VEGF₁₂₁ sequence 2 GACAAGAAAATCCCTGTGG 27Splice variant VEGF₁₆₅ sequence 3 GAAAATCCCTGTGGGCCTT 28Splice variant VEGF₁₈₉ sequence 4 AATCCCTGTGGGCCTTGCT 29Splice variant VEGF₂₀₆ sequence 5 TCCCTGTGGGCCTTGCTCA 30TCATCACGAAGTGGTGAAG 8 GCATTTGTTTGTACAAGAT 31 UCAUCACGAAGUGGUGAAGUU 9GATCCGCAGACGTGTAAAT 32 UUAGUAGUGCUUCACCACUUC 10 ATGTTCCTGCAAAAACACA 33UCAUCACGAAGUGGUGAAGTT 11 TGTTCCTGCAAAAACACAG 34 TTAGUAGUGCUUCACCACUUC 12AAACACAGACTCGCGTTGC 35 AACGTACTTGCAGATGTGACA 13 AACACAGACTCGCGTTGCA 36GTTCATGGATGTCTATCAG 14 ACACAGACTCGCGTTGCAA 37 TCGAGACCCTGGTGGACAT 15CACAGACTCGCGTTGCAAG 38 TGACGAGGGCCTGGAGTGT 16 GGCGAGGCAGCTTGAGTTA 39TGACGAGGGCCTGGAGTGT 17 ACGAACGTACTTGCAGATG 40 CATCACCATGCAGATTATG 18CGAACGTACTTGCAGATGT 41 ACCTCACCAAGGCCAGCAC 19 CGTACTTGCAGATGTGACA 42GGCCAGCACATAGGAGAGA 20 GTGGTCCCAGGCTGCACCC 43 CAAATGTGAATGCAGACCA 21GGAGGAGGGCAGAATCATC 44 ATGTGAATGCAGACCAAAG 22 GTGGTGAAGTTCATGGATG 45TGCAGACCAAAGAAAGATA 23 AATCATCACGAAGTGGTGAAG 46 AGAAAGATAGAGCAAGACA 24AAGTTCATGGATGTCTATCAG 47 GAAAGATAGAGCAAGACAA 25 AATCGAGACCCTGGTGGACAT 48AATGACGAGGGCCTGGAGTGT 49 AATGTTCCTGCAAAAACACAGAC 65AACATCACCATGCAGATTATG 50 AAAAACACAGACTCGCGTTGCAA 66AAACCTCACCAAGGCCAGCAC 51 AAAACACAGACTCGCGTTGCAAG 67AAGGCCAGCACATAGGAGAGA 52 AAACACAGACTCGCGTTGCAAGG 68AACAAATGTGAATGCAGACCA 53 AACACAGACTCGCGTTGCAAGGC 69AAATGTGAATGCAGACCAAAG 54 AAGGCGAGGCAGCTTGAGTTAAA 70AATGCAGACCAAAGAAAGATA 55 AAACGAACGTACTTGCAGATGTG 71AAAGAAAGATAGAGCAAGACA 56 AACGAACGTACTTGCAGATGTGA 72AAGAAAGATAGAGCAAGACAA 57 AAGTGGTCCCAGGCTGCACCCAT 73AAGATAGAGCAAGACAAGAAAAT 58 AAGGAGGAGGGCAGAATCATCAC 74AAGACAAGAAAATCCCTGTGGGC 59 AAGTGGTGAAGTTCATGGATGTC 75AAGAAAATCCCTGTGGGCCTTGC 60 AAAATCCCTGTGGGCCTTGCTCA 76AATCCCTGTGGGCCTTGCTCAGA 61 ACCUCACCAAGGCCAGCACTT 77AAGCATTTGTTTGTACAAGATCC 62 GUGCUGGCCUUGGUGAGGUTT 78AAGATCCGCAGACGTGTAAATGT 63 GGCTACGTCCAGCGCACC 79 AAATGTTCCTGCAAAAACACAGA64 AAACCUCACCAAAGCCAGCAC 80 ACCUCACCAAGGCCAGCAC 119 GUGCUGGCCUUGGUGAGGU120

TABLE 2 VEGF Target Sequences selectively excluding VEGF₁₆₅b siRNA NameTarget sequence (5′-3′) OPK-HVB-001 AACGTACTTGCAGATGTGA (SEQ ID NO: 86)OPK-HVB-002 ACGTACTTGCAGATGTGAC (SEQ ID NO: 87) OPK-HVB-003CGTACTTGCAGATGTGACA (SEQ ID NO: 42) OPK-HVB-004GTACTTGCAGATGTGACAA (SEQ ID NO: 88) OPK-HVB-005TACTTGCAGATGTGACAAG (SEQ ID NO: 89) OPK-HVB-006ACTTGCAGATGTGACAAGC (SEQ ID NO: 90) OPK-HVB-007CTTGCAGATGTGACAAGCC (SEQ ID NO: 91) OPK-HVB-008TTGCAGATGTGACAAGCCG (SEQ ID NO: 92) OPK-HVB-009TGCAGATGTGACAAGCCGA (SEQ ID NO: 93) OPK-HVB-010GCAGATGTGACAAGCCGAG (SEQ ID NO: 94) OPK-HVB-011CAGATGTGACAAGCCGAGG (SEQ ID NO: 95) OPK-HVB-012AGATGTGACAAGCCGAGGC (SEQ ID NO: 96) OPK-HVB-013GATGTGACAAGCCGAGGCG (SEQ ID NO: 97) OPK-HVB-014ATGTGACAAGCCGAGGCGG (SEQ ID NO: 98) OPK-HVB-004beGTACTTGCAGATGTGACAA (SEQ ID NO: 99) OPK-HVB-009beTGCAGATGTGACAAGCCGA (SEQ ID NO: 100) OPK-HVB-010beGCAGATGTGACAAGCCGAG (SEQ ID NO: 101) OPK-HVB-012beAGATGTGACAAGCCGAGGC (SEQ ID NO: 102) OPK-HVB-001aAACGTACTTGCAGATGT (SEQ ID NO: 103) OPK-HVB-002aACGTACTTGCAGATGTG (SEQ ID NO: 104) OPK-HVB-003aCGTACTTGCAGATGTGA (SEQ ID NO: 105) OPK-HVB-004aGTACTTGCAGATGTGAC (SEQ ID NO: 106) OPK-HVB-005aTACTTGCAGATGTGACA (SEQ ID NO: 107) OPK-HVB-006aACTTGCAGATGTGACAA (SEQ ID NO: 108) OPK-HVB-007aCTTGCAGATGTGACAAG (SEQ ID NO: 109) OPK-HVB-008aTTGCAGATGTGACAAGC (SEQ ID NO: 110) OPK-HVB-009aTGCAGATGTGACAAGCC (SEQ ID NO: 111) OPK-HVB-010aGCAGATGTGACAAGCCG (SEQ ID NO: 112) OPK-HVB-011aCAGATGTGACAAGCCGA (SEQ ID NO: 113) OPK-HVB-012aAGATGTGACAAGCCGAG (SEQ ID NO: 114) OPK-HVB-013aGATGTGACAAGCCGAGG (SEQ ID NO: 115) OPK-HVB-014aATGTGACAAGCCGAGGC (SEQ ID NO: 116) OPK-HVB-015aTGTGACAAGCCGAGGCG (SEQ ID NO: 117) OPK-HVB-016aGTGACAAGCCGAGGCGG (SEQ ID NO: 118) The sequences with the names“OPK-HVB-XXXbe” refer to sequences that are 19 mer blunt endcounterparts of the similar 21 mers. The sequences with the names“OPVHVB-XXXa” refer to 19 mers where there is a 17 bp nucleotidesequence with a dTdT overhang. Other sequences not specificallyexemplified herein but targeting VEGF while sparing VEGF165b can also bemade with similar properties.

Other blunt end nucleic acid molecules can also be used, but that do notnecessarily spare VEGF165b. For example, an siRNA comprising a sensestrand SEQ ID NO: 119 and an antisense strand comprising SEQ ID NO: 120can be used. An siRNA comprising SEQ ID NO: 119 and SEQ ID NO: 120,wherein each siRNA comprises blunt ends can also be referred to asbevasiranib-be. For example, in some embodiments, the siRNA is a 19merwith a blunt ends comprising SEQ ID NO: 119 and SEQ ID NO: 120 (See FIG.36).

The siRNA can be obtained using a number of techniques known to those ofskill in the art. For example, the siRNA can be chemically synthesizedor recombinantly produced using methods known in the art, such as theDrosophila in vitro system described in U.S. published application2002/0086356 of Tuschl et al., the entire disclosure of which is hereinincorporated by reference.

In certain embodiments, the siRNA are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. The siRNA can be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

Alternatively, siRNA can also be expressed from recombinant circular orlinear DNA plasmids using any suitable promoter. Suitable promoters forexpressing siRNA from a plasmid include, for example, the U6 or H1 RNApol III promoter sequences and the cytomegalovirus promoter. Selectionof other suitable promoters is within the skill in the art. Therecombinant plasmids of the invention can also comprise inducible orregulatable promoters for expression of the siRNA in a particular tissueor in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolatedfrom cultured cell expression systems by standard techniques, or can beexpressed intracellularly at or near the area of neovascularization invivo. The use of recombinant plasmids to deliver siRNA to cells in vivois discussed in more detail below.

siRNA can be expressed from a recombinant plasmid either as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions.

Selection of plasmids suitable for expressing siRNA, methods forinserting nucleic acid sequences for expressing the siRNA into theplasmid, and methods of delivering the recombinant plasmid to the cellsof interest are within the skill in the art. See, for example Tuschl, T.(2002), Nat. Biotechnol, 20: 446-448; Brummelkamp T R et al. (2002),Science 296: 550-553; Miyagishi M et al. (2002), Nat. Biotechnol. 20:497-500; Paddison P J et al. (2002), Genes Dev. 16: 948-958; Lee N S etal. (2002), Nat. Biotechnol. 20: 500-505; and Paul C P et al. (2002),Nat. Biotechnol. 20: 505-508, the entire disclosures of which are hereinincorporated by reference.

A plasmid comprising nucleic acid sequences for expressing an siRNA isdescribed in Example 7 below. That plasmid, called pAAVsiRNA, comprisesa sense RNA strand coding sequence in operable connection with a polyTtermination sequence under the control of a human U6 RNA promoter, andan antisense RNA strand coding sequence in operable connection with apolyT termination sequence under the control of a human U6 RNA promoter.The plasmid pAAVsiRNA is ultimately intended for use in producing anrecombinant adeno-associated viral vector comprising the same nucleicacid sequences for expressing an siRNA.

As used herein, “in operable connection with a polyT terminationsequence” means that the nucleic acid sequences encoding the sense orantisense strands are immediately adjacent to the polyT terminationsignal in the 5′ direction. During transcription of the sense orantisense sequences from the plasmid, the polyT termination signals actto terminate transcription.

As used herein, “under the control” of a promoter means that the nucleicacid sequences encoding the sense or antisense strands are located 3′ ofthe promoter, so that the promoter can initiate transcription of thesense or antisense coding sequences.

The siRNA can also be expressed from recombinant viral vectorsintracellularly at or near the area of neovascularization in vivo. Therecombinant viral vectors of the invention comprise sequences encodingthe siRNA and any suitable promoter for expressing the siRNA sequences.Suitable promoters include, for example, the U6 or H1 RNA pol IIIpromoter sequences and the cytomegalovirus promoter. Selection of othersuitable promoters is within the skill in the art. The recombinant viralvectors of the invention can also comprise inducible or regulatablepromoters for expression of the siRNA in a particular tissue or in aparticular intracellular environment. The use of recombinant viralvectors to deliver siRNA to cells in vivo is discussed in more detailbelow.

siRNA can be expressed from a recombinant viral vector either as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions.

Any viral vector capable of accepting the coding sequences for the siRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of the viral vectors can also be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses. For example, an AAV vector of the inventioncan be pseudotyped with surface proteins from vesicular stomatitis virus(VSV), rabies, Ebola, Mokola, and the like.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;and Anderson W F (1998), Nature 392: 25-30, the entire disclosures ofwhich are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the siRNA is expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector comprising, for example, either the U6 or H1 RNA promoters,or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the siRNA, a method for constructingthe recombinant AV vector, and a method for delivering the vector intotarget cells, are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010.

Suitable AAV vectors for expressing the siRNA, methods for constructingthe recombinant AAV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol., 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S.Pat. No. 5,139,941; International Patent Application No. WO 94/13788;and International Patent Application No. WO 93/24641, the entiredisclosures of which are herein incorporated by reference. An exemplarymethod for generating a recombinant AAV vector of the invention isdescribed in Example 7 below.

The ability of an siRNA containing a given target sequence to causeRNAi-mediated degradation of the target mRNA can be evaluated usingstandard techniques for measuring the levels of RNA or protein in cells.For example, siRNA can be delivered to cultured cells, and the levels oftarget mRNA can be measured by Northern blot or dot blotting techniques,or by quantitative RT-PCR. Alternatively, the levels of VEGF and itsisoforms as well as Flt-1 or Flk-1/KDR receptor protein in the culturedcells can be measured by ELISA or Western blot. A suitable cell culturesystem for measuring the effect of the present siRNA on target mRNA orprotein levels is described in Example 1 below.

RNAi-mediated degradation of target mRNA by an siRNA containing a giventarget sequence can also be evaluated with animal models ofneovascularization, such as the ROP or CNV mouse models. For example,areas of neovascularization in an ROP or CNV mouse can be measuredbefore and after administration of an siRNA and, in some embodiments,compared to an untreated animal. A reduction in the areas ofneovascularization in these models upon administration of the siRNAindicates, in some embodiments, the down-regulation of the target mRNA(see Example 6 below).

As discussed above, the siRNA is capable of targeting and causing theRNAi-mediated degradation of VEGF and its isoforms as well as Flt-1 orFlk-1/KDR mRNA, or alternative splice forms, mutants or cognatesthereof, preferably VEGF, and more preferably human VEGF. Degradation ofthe target mRNA by the present siRNA reduces the production of afunctional gene product from the VEGF and its isoforms as well as Flt-1or Flk-1/KDR genes. Thus, another embodiment of the present inventionprovides a method of inhibiting expression of VEGF and its isoforms,such as VEGF₁₂₁(SEQ ID NO: 2), VEGF₁₆₅ (SEQ ID NO: 3), and VEGF₁₈₉(SEQID NO: 4), VEGF₂₀₆(SEQ ID NO: 5; GenBank Accession No. CS245579),VEGF₁₈₃ (GenBank Accession No. AJ010438), VEGF₁₄₈ (GenBank Accession No.AF091352), and/or VEGF₁₄₅ (GenBank Accession No. CS245578), as well asFlt-1 or Flk-1/KDR in a subject, comprising administering an effectiveamount of an siRNA to the subject, such that the target mRNA isdegraded. As the products of the VEGF and its isoforms as well as Flt-1and Flk-1/KDR genes are required for initiating and maintainingangiogenesis, another embodiment of the present invention provides amethod of inhibiting angiogenesis in a subject by the RNAi-mediateddegradation of the target mRNA by the present siRNA.

RNAi-mediated degradation of the target mRNA can be detected bymeasuring levels of the target mRNA or protein in the cells of asubject, using standard techniques for isolating and quantifying mRNA orprotein as described above.

Inhibition of angiogenesis can be evaluated by directly measuring theprogress of pathogenic or nonpathogenic angiogenesis in a subject; forexample, by observing the size of a neovascularized area before andafter treatment with the siRNA. An inhibition of angiogenesis isindicated if the size of the neovascularized area stays the same or isreduced. Techniques for observing and measuring the size ofneovascularized areas in a subject are within the skill in the art; forexample, areas of choroid neovascularization can be observed, forexample, by fluorescein angiography.

Inhibition of angiogenesis can also be inferred through observing achange or reversal in a pathogenic condition associated with theangiogenesis. For example, in ARMD, a slowing, halting or reversal ofvision loss indicates an inhibition of angiogenesis in the choroid. Fortumors, a slowing, halting or reversal of tumor growth, or a slowing orhalting of tumor metastasis, indicates an inhibition of angiogenesis ator near the tumor site Inhibition of non-pathogenic angiogenesis canalso be inferred from, for example, fat loss or a reduction incholesterol levels upon administration of the siRNA.

It is understood that the siRNA can degrade the target mRNA (and thusinhibit angiogenesis) in substoichiometric amounts. Without wishing tobe bound by any theory, it is believed that the siRNA causes degradationof the target mRNA in a catalytic manner. Thus, compared to standardanti-angiogenic therapies, significantly less siRNA needs to bedelivered at or near the site of neovascularization to have atherapeutic effect.

One skilled in the art can readily determine an effective amount of thesiRNA to be administered to a given subject, by taking into accountfactors such as the size and weight of the subject; the extent of theneovascularization or disease penetration; the age, health and sex ofthe subject; the route of administration; and whether the administrationis regional or systemic. Generally, an effective amount of the siRNAcomprises an intercellular concentration at or near theneovascularization site of from about 1 nanomolar (nM) to about 100 nM,preferably from about 2 nM to about 50 nM, more preferably from about2.5 nM to about 10 nM. It is contemplated that greater or lesser amountsof siRNA can be administered.

The present methods can be used to inhibit angiogenesis which isnon-pathogenic; i.e., angiogenesis which results from normal processesin the subject. Examples of non-pathogenic angiogenesis includeendometrial neovascularization, and processes involved in the productionof fatty tissues or cholesterol. Thus, the invention provides a methodfor inhibiting non-pathogenic angiogenesis, e.g., for controlling weightor promoting fat loss, for reducing cholesterol levels, or as anabortifacient.

The present methods can also inhibit angiogenesis which is associatedwith an angiogenic disease; i.e., a disease in which pathogenicity isassociated with inappropriate or uncontrolled angiogenesis. For example,most cancerous solid tumors generate an adequate blood supply forthemselves by inducing angiogenesis in and around the tumor site. Thistumor-induced angiogenesis is often required for tumor growth, and alsoallows metastatic cells to enter the bloodstream.

Other angiogenic diseases include diabetic retinopathy, age-relatedmacular degeneration (ARMD), psoriasis, rheumatoid arthritis and otherinflammatory diseases. These diseases are characterized by thedestruction of normal tissue by newly formed blood vessels in the areaof neovascularization. For example, in ARMD, the choroid is invaded anddestroyed by capillaries. The angiogenesis-driven destruction of thechoroid in ARMD eventually leads to partial or full blindness.

Preferably, an siRNA is used to inhibit the growth or metastasis ofsolid tumors associated with cancers; for example breast cancer, lungcancer, head and neck cancer, brain cancer, abdominal cancer, coloncancer, colorectal cancer, esophagus cancer, gastrointestinal cancer,glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma,ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma,Wilm's tumor, multiple myeloma; skin cancer (e.g., melanoma), lymphomasand blood cancer.

More preferably, an siRNA is used to inhibit choroidalneovascularization in age-related macular degeneration.

For treating angiogenic diseases, the siRNA can administered to asubject in combination with a pharmaceutical agent which is differentfrom the present siRNA. Alternatively, the siRNA can be administered toa subject in combination with another therapeutic method designed totreat the angiogenic disease. For example, the siRNA can be administeredin combination with therapeutic methods currently employed for treatingcancer or preventing tumor metastasis (e.g., radiation therapy,chemotherapy, and surgery). For treating tumors, the siRNA is preferablyadministered to a subject in combination with radiation therapy, or incombination with chemotherapeutic agents such as cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

In the present methods, the present siRNA can be administered to thesubject either as naked siRNA, in conjunction with a delivery reagent,or as a recombinant plasmid or viral vector which expresses the siRNA.

Suitable delivery reagents for administration in conjunction with thepresent siRNA include, but not limited to, the Minis Transit TKOlipophilic reagent; lipofectin; lipofectamine; cellfectin; orpolycations (e.g., polylysine), or liposomes. In some embodiments thedelivery reagent is RiboJuice™ (Novagen), a siRNA transfection reagent,which comprises amine and lipid based reagents. A preferred deliveryreagent is a liposome. In some embodiments, the siRNA is delivered freeof a liposomal delivery agent.

Liposomes can aid in the delivery of the siRNA to a particular tissue,such as retinal or tumor tissue, and can also increase the bloodhalf-life of the siRNA. Liposomes suitable for use in the invention areformed from standard vesicle-forming lipids, which generally includeneutral or negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. A variety of methods are known forpreparing liposomes, for example as described in Szoka et al. (1980),Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which areherein incorporated by reference.

Preferably, the liposomes encapsulating the present siRNA comprises aligand molecule that can target the liposome to a particular cell ortissue at or near the site of angiogenesis. Ligands which bind toreceptors prevalent in tumor or vascular endothelial cells, such asmonoclonal antibodies that bind to tumor antigens or endothelial cellsurface antigens, are preferred.

Particularly preferably, the liposomes encapsulating the present siRNAare modified so as to avoid clearance by the mononuclear macrophage andreticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer which significantly decreases the uptakeof the liposomes by the macrophage-monocyte system (“MMS”) andreticuloendothelial system (“RES”); e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is herein incorporated byreference. Liposomes modified with opsonization-inhibition moieties thusremain in the circulation much longer than unmodified liposomes. Forthis reason, such liposomes are sometimes called “stealth” liposomes.

Stealth liposomes are known to accumulate in tissues fed by porous or“leaky” microvasculature. Thus, target tissue characterized by suchmicrovasculature defects, for example solid tumors, will efficientlyaccumulate these liposomes; see Gabizon, et al. (1988), P.N.A.S., USA,18: 6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation inthe liver and spleen. Thus, liposomes of the invention that are modifiedwith opsonization-inhibition moieties can deliver the present siRNA totumor cells.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM_(i). Copolymersof PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginicacid, carrageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Recombinant plasmids which express siRNA are discussed above. Suchrecombinant plasmids can also be administered directly or in conjunctionwith a suitable delivery reagent, including the Mirus Transit LT1lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations(e.g., polylysine) or liposomes. Recombinant viral vectors which expresssiRNA are also discussed above, and methods for delivering such vectorsto an area of neovascularization in a patient are within the skill inthe art.

The siRNA can be administered to the subject by any means suitable fordelivering the siRNA to the cells of the tissue at or near the area ofneovascularization. For example, the siRNA can be administered by genegun, electroporation, or by other suitable parenteral or enteraladministration routes.

Suitable enteral administration routes include oral, rectal, orintranasal delivery.

Suitable parenteral administration routes include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissueadministration (e.g., peri-tumoral and intra-tumoral injection,intra-retinal injection or subretinal injection); subcutaneous injectionor deposition including subcutaneous infusion (such as by osmoticpumps); direct (e.g., topical) application to the area at or near thesite of neovascularization, for example by a catheter or other placementdevice (e.g., a corneal pellet or a suppository, eye-dropper, or animplant comprising a porous, non-porous, or gelatinous material); andinhalation. Suitable placement devices include the ocular implantsdescribed in U.S. Pat. Nos. 5,902,598 and 6,375,972, and thebiodegradable ocular implants described in U.S. Pat. No. 6,331,313, theentire disclosures of which are herein incorporated by reference. Suchocular implants are available from Control Delivery Systems, Inc.(Watertown, Mass.) and Oculex Pharmaceuticals, Inc. (Sunnyvale, Calif.).

In a preferred embodiment, injections or infusions of the siRNA aregiven at or near the site of neovascularization. More preferably, thesiRNA is administered topically to the eye, e.g. in liquid or gel formto the lower eye lid or conjunctival cul-de-sac, as is within the skillin the art (see, e.g., Acheampong A A et al, 2002, Drug Metabol. andDisposition 30: 421-429, the entire disclosure of which is hereinincorporated by reference).

Typically, the siRNA is administered topically to the eye in amounts offrom about 5 microliters to about 75 microliters, for example from about7 microliters to about 50 microliters, preferably from about 10microliters to about 30 microliters. It is understood that topicalinstillation in the eye of siRNA in volumes greater than 75 microliterscan result in loss of siRNA from the eye through spillage and drainage.Thus, it is preferable to administer a high concentration of siRNA(e.g., 100-1000 nM) in as small a volume as possible.

A particularly preferred parenteral administration route is intraocularadministration. It is understood that intraocular administration of thepresent siRNA can be accomplished by injection or direct (e.g., topical)administration to the eye, as long as the administration route allowsthe siRNA to enter the eye. In addition to the topical routes ofadministration to the eye described above, suitable intraocular routesof administration include intravitreal, intraretinal, subretinal,subtenon, peri- and retro-orbital, trans-corneal and trans-scleraladministration. Such intraocular administration routes are within theskill in the art; see, e.g., and Acheampong A A et al, 2002, supra; andBennett et al. (1996), Hum. Gene Ther. 7: 1763-1769 and Ambati J et al.,2002, Progress in Retinal and Eye Res. 21: 145-151, the entiredisclosures of which are herein incorporated by reference. In anotherpreferred embodiment, the siRNA is administered by intravitrealinjection.

The siRNA can be administered in a single dose or in multiple doses.Where the administration of the siRNA is by infusion, the infusion canbe a single sustained dose or can be delivered by multiple infusions.Injection of the agent directly into the tissue is at or near the siteof neovascularization preferred. Multiple injections of the agent intothe tissue at or near the site of neovascularization are particularlypreferred.

One skilled in the art can also readily determine an appropriate dosageregimen for administering the siRNA to a given subject. For example, thesiRNA can be administered to the subject once, such as by a singleinjection or deposition at or near the neovascularization site.Alternatively, the siRNA can be administered to a subject multiple timesdaily or weekly. For example, the siRNA can be administered to a subjectonce weekly for a period of from about three to about twenty-eightweeks, and alternatively from about seven to about ten weeks. In acertain dosage regimen, the siRNA is injected at or near the site ofneovascularization (e.g., intravitreally) once a week for seven weeks.It is understood that periodic administrations of the siRNA for anindefinite length of time may be necessary for subjects suffering from achronic neovascularization disease, such as wet ARMD or diabeticretinopathy.

Where a dosage regimen comprises multiple administrations, it isunderstood that the effective amount of siRNA administered to thesubject can comprise the total amount of siRNA administered over theentire dosage regimen.

The siRNA are preferably formulated as pharmaceutical compositions priorto administering to a subject, according to techniques known in the art.Pharmaceutical compositions of the present invention are characterizedas being at least sterile and pyrogen-free. As used herein,“pharmaceutical formulations” include formulations for human andveterinary use. Methods for preparing pharmaceutical compositions of theinvention are within the skill in the art, for example as described inRemington's Pharmaceutical Science, 17th ed., Mack Publishing Company,Easton, Pa. (1985), the entire disclosure of which is hereinincorporated by reference.

In one embodiment, the pharmaceutical formulations comprise an siRNA(e.g., 0.1 to 90% by weight), or a physiologically acceptable saltthereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, saline solutions (e.g., normal saline or balanced salinesolutions such as Hank's or Earle's balanced salt solutions), 0.4%saline, 0.3% glycine, hyaluronic acid and the like.

Pharmaceutical compositions can also comprise conventionalpharmaceutical excipients and/or additives. Suitable pharmaceuticalexcipients include stabilizers, antioxidants, osmolality adjustingagents, buffers, and pH adjusting agents. Suitable additives includephysiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized

For topical administration to the eye, conventional intraocular deliveryreagents can be used. For example, pharmaceutical compositions of theinvention for topical intraocular delivery can comprise saline solutionsas described above, corneal penetration enhancers, insoluble particles,petrolatum or other gel-based ointments, polymers which undergo aviscosity increase upon instillation in the eye, or mucoadhesivepolymers. Preferably, the intraocular delivery reagent increases cornealpenetration, or prolongs preocular retention of the siRNA throughviscosity effects or by establishing physicochemical interactions withthe mucin layer covering the corneal epithelium.

Suitable insoluble particles for topical intraocular delivery includethe calcium phosphate particles described in U.S. Pat. No. 6,355,271 ofBell et al., the entire disclosure of which is herein incorporated byreference. Suitable polymers which undergo a viscosity increase uponinstillation in the eye include polyethylenepolyoxypropylene blockcopolymers such as poloxamer 407 (e.g., at a concentration of 25%),cellulose acetophthalate (e.g., at a concentration of 30%), or alow-acetyl gellan gum such as Gelrite® (available from CP Kelco,Wilmington, Del.). Suitable mucoadhesive polymers include hydrocolloidswith multiple hydrophilic functional groups such as carboxyl, hydroxyl,amide and/or sulfate groups; for example, hydroxypropylcellulose,polyacrylic acid, high-molecular weight polyethylene glycols(e.g., >200,000 number average molecular weight), dextrans, hyaluronicacid, polygalacturonic acid, and xylocan. Suitable corneal penetrationenhancers include cyclodextrins, benzalkonium chloride, polyoxyethyleneglycol lauryl ether (e.g., Brij® 35), polyoxyethylene glycol stearylether (e.g., Brij® 78), polyoxyethylene glycol oleyl ether (e.g., Brij®98), ethylene diamine tetraacetic acid

(EDTA), digitonin, sodium taurocholate, saponins and polyoxyethylatedcastor oil such as Cremaphor EL.

For solid compositions, conventional nontoxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of one or more siRNA. A pharmaceutical compositionfor aerosol (inhalational) administration can comprise 0.01-20% byweight, preferably 1%-10% by weight, of one or more siRNA encapsulatedin a liposome as described above, and propellant. A carrier can also beincluded as desired; e.g., lecithin for intranasal delivery.

The invention will now be illustrated with the following non-limitingexamples.

Example 1 siRNA Transfection and Hypoxia Induction In Vitro

siRNA Design—A 19 nt sequence located 329 nt from the 5′ end of humanVEGF mRNA was chosen as a target sequence: AAACCTCACCAAGGCCAGCAC (SEQ IDNO: 51). To ensure that it was not contained in the mRNA from any othergenes, this target sequence was entered into the BLAST search engineprovided by NCBI. The use of the BLAST algorithm is described inAltschul et al. (1990), J. Mol. Biol. 215: 403-410 and Altschul et al.(1997), Nucleic Acids Res. 25: 3389-3402, the disclosures of which areherein incorporated by reference in their entirety. As no other mRNA wasfound which contained the target sequence, an siRNA duplex wassynthesized to target this sequence (Dharmacon Research, Inc.,Lafayette, Colo.).

The siRNA duplex had the following sense and antisense strands.

sense: (SEQ ID NO: 77) 5′-accucaccaaggccagcacTT-3′. antisense:(SEQ ID NO: 78) 5′-gugcuggccuuggugagguTT-3′.

Together, the siRNA sense and antisense strands formed a 19 ntdouble-stranded siRNA with TT 3′ overhangs (shown in bold) on eachstrand. This siRNA was termed “Candidate 5” or “Cand5.” Other siRNAwhich target human VEGF mRNA were designed and tested as described forCand5 (bevasiranib).

An siRNA targeting the following sequence in green fluorescent protein(GFP) mRNA was used as a nonspecific control: GGCTACGTCCAGCGCACC (SEQ IDNO: 79). The siRNA was purchased from Dharmacon (Lafayette, Colo.).

siRNA Transfection and Hypoxia Induction In Vitro—Human cell lines (293;Hela and ARPE19) were separately seeded into 24-well plates in 250microliters of complete DMEM medium one day prior to transfection, sothat the cells were ˜50% confluent at the time of transfection. Cellswere transfected with 2.5 nM Cand5 siRNA, and with either no siRNA or2.5 nM non-specific siRNA (targeting GFP) as controls. Transfectionswere performed in all cell lines with the “Transit TKO Transfection”reagent, as recommended by the manufacturer (Mirus).

Twenty four hours after transfection, hypoxia was induced in the cellsby the addition of deferoxamine mesylate to a final concentration of 130micromolar in each well. Twenty four hours post-transfection, the cellculture medium was removed from all wells, and a human VEGF ELISA (R&Dsystems, Minneapolis, Minn.) was performed on the culture medium asdescribed in the Quantikine human VEGF ELISA protocol available from themanufacturer, the entire disclosure of which is herein incorporated byreference.

As can be seen in FIG. 1, RNAi degradation induced by Candy siRNAsignificantly reduces the concentration of VEGF produced by the hypoxic293 and HeLa cells. There was essentially no difference in the amount ofVEGF produced by hypoxic cells treated with either no siRNA or thenon-specific siRNA control. Similar results were also seen with humanARPE19 cells treated under the same conditions. Thus, RNA interferencewith VEGF-targeted siRNA disrupts the pathogenic up-regulation of VEGFin human cultured cells in vitro.

The experiment outlined above was repeated on mouse NIH 3T3 cells usinga mouse-specific VEGF siRNA (see Example 6 below), and VEGF productionwas quantified with a mouse VEGF ELISA (R&D systems, Minneapolis, Minn.)as described in the Quantikine mouse VEGF ELISA protocol available fromthe manufacturer, the entire disclosure of which is herein incorporatedby reference. Results similar to those reported in FIG. 1 for the humancell lines were obtained.

Example 2 Effect of Increasing siRNA Concentration on VEGF Production inHuman Cultured Cells

The experiment outlined in Example 1 was repeated with human 293, HeLaand ARPE19 cells using a range of siRNA concentrations from 10 nM to 50nM. The ability of the Cand5 siRNA to down-regulate VEGF productionincreased moderately up to approximately 13 nM siRNA, but a plateaueffect was seen above this concentration. These results highlight thecatalytic nature of siRNA-mediated RNAi degradation of mRNA, as theplateau effect appears to reflect VEGF production from the few cells nottransfected with the siRNA. For the majority of cells which had beentransfected with the siRNA, the increased VEGF mRNA production inducedby the hypoxia is outstripped by the siRNA-induced degradation of thetarget mRNA at siRNA concentrations greater than about 13 nM.

Example 3 Specificity of siRNA Targeting

NIH 3T3 mouse fibroblasts were grown in 24-well plates under standardconditions, so that the cells were ˜50% confluent one day prior totransfection. The human VEGF siRNA Cand5 was transfected into a NIH 3T3mouse fibroblasts as in Example 1. Hypoxia was then induced in thetransfected cells, and murine VEGF concentrations were measured by ELISAas in Example 1.

The sequence targeted by the human VEGF siRNA Cand5 differs from themurine VEGF mRNA by one nucleotide. As can be seen in FIG. 2, the humanVEGF siRNA has no affect on the ability of the mouse cells toup-regulate mouse VEGF after hypoxia. These results show that siRNAinduced RNAi degradation is sequence-specific to within a one nucleotideresolution.

Example 4 In Vivo delivery of siRNA to Murine Retinal Pigment EpithelialCells

VEGF is upregulated in the retinal pigment epithelial (RPE) cells ofhuman patients with age-related macular degeneration (ARMD). To showthat functional siRNA can be delivered to RPE cells in vivo, GFP wasexpressed in mouse retinas with a recombinant adenovirus, and GFPexpression was silenced with siRNA. The experiment was conducted asfollows.

One eye from each of five adult C57/Black6 mice (Jackson Labs, BarHarbor, Me.) was injected subretinally as described in Bennett et al.(1996), supra., with a mixture containing ˜1×10⁸ particles of adenoviruscontaining eGFP driven by the CMV promoter and 20 picomoles of siRNAtargeting eGFP conjugated with transit TKO reagent (Mirus).

As positive control, the contralateral eyes were injected with a mixturecontaining ˜1×10⁸ particles of adenovirus containing eGFP driven by theCMV promoter and 20 picomoles of siRNA targeting human VEGF conjugatedwith transit TKO reagent (Mirus). Expression of GFP was detected byfundus ophthalmoscopy 48 hours and 60 hours after injection. Animalswere sacrificed at either 48 hours or 60 hours post-injection. The eyeswere enucleated and fixed in 4% paraformaldehyde, and were preparedeither as flat mounts or were processed into 10 micron cryosections forfluorescent microscopy.

No GFP fluorescence was detectable by ophthalmoscopy in the eyes whichreceived the siRNA targeted to GFP mRNA in 4 out of 5 mice, whereas GFPfluorescence was detectable in the contralateral eye which received thenon-specific control siRNA. A representative flat mount analyzed byfluorescence microscopy showed a lack of GFP fluorescence in the eyewhich received GFP siRNA, as compared to an eye that received thenon-specific control siRNA. Cryosections of another retina showed thatthe recombinant adenovirus efficiently targets the RPE cells, and whenthe adenovirus is accompanied by siRNA targeted to GFP mRNA, expressionof the GFP transgene is halted.

While there is some GFP fluorescence detectable by fluorescencemicroscopy in eyes that received siRNA targeted to GFP mRNA, thefluorescence is greatly suppressed as compared to controls that receivednon-specific siRNA. These data demonstrate that functional siRNA can bedelivered in vivo to RPE cells.

Example 5 In Vivo Expression and siRNA-Induced RNAi Degradation of HumanVEGF in Murine Retinas

In order to demonstrate that siRNA targeted to VEGF functioned in vivo,an exogenous human VEGF expression cassette was delivered to mouse RPEcells via an adenovirus by subretinal injection, as in Example 4. Oneeye received Cand5 siRNA, and the contralateral eye received siRNAtargeted to GFP mRNA. The animals were sacrificed 60 hourspost-injection, and the injected eyes were removed and snap frozen inliquid N₂ following enucleation. The eyes were then homogenized in lysisbuffer, and total protein was measured using a standard Bradford proteinassay (Roche, Germany). The samples were normalized for total proteinprior to assaying for human VEGF by ELISA as described in Example 1.

The expression of VEGF was somewhat variable from animal to animal. Thevariability of VEGF levels correlated well to those observed in the GFPexperiments of Example 4, and can be attributed to some error frominjection to injection, and the differential ability of adenovirus todelivery the target gene in each animal. However, there was asignificant attenuation of VEGF expression in each eye that receivedVEGF siRNA, as compared to the eyes receiving the non-specific controlsiRNA (FIG. 4). These data indicate that the Cand5 siRNA was potent andeffective in silencing human VEGF expression in murine RPE cells invivo.

Example 6 Inhibition of Choroidal Neovascularization in the Mouse CNVModel

There is evidence that choroidal neovascularization in ARMD is due tothe upregulation of VEGF in the RPE cells. This human pathologiccondition can be modeled in the mouse by using a laser to burn a spot onthe retina (“laser photo-coagulation” or “laser induction”). During thehealing process, VEGF is believed to be up-regulated in the RPE cells ofthe burned region, leading to re-vascularization of the choroid. Thismodel is called the mouse choroidal neovascularization (“CNV”) model.

For rescue of the mouse CNV model, a mouse siRNA was designed thatincorporated a one nucleotide change from the human “Cand5” siRNA fromExample 1. The mouse siRNA specifically targeted mouse VEGF mRNA at thesequence AAACCUCACCAAAGCCAGCAC (SEQ ID NO: 80). Other siRNA that targetmouse VEGF were also designed and tested. The GFP siRNA used as anonspecific control in Example 1 was also used as a non-specific controlhere.

Twenty four hours after laser induction, one eye from each of elevenadult C57/Black6 mice (Jackson Labs, Bar Harbor, Me.) was injectedsubretinally with a mixture containing ˜1×10⁸ particles of adenoviruscontaining LacZ driven by the CMV promoter and 20 picomoles of siRNAtargeting mouse VEGF conjugated with transit TKO reagent (Mirus), as inExample 4. As a control, contralateral eyes received a mixturecontaining ˜1×10⁸ particles of adenovirus containing LacZ driven by theCMV promoter and 20 picomoles of siRNA targeting GFP conjugated withtransit TKO reagent (Mirus).

Fourteen days after the laser treatment, the mice were perfused withfluorescein and the area of neovascularization was measured around theburn spots. Areas of the burn spots in the contra-lateral eye were usedas a control. The site of neovascularization around the burn spots inanimals that received siRNA targeting mouse VEGF was, on average, 1/4the area of the control areas. These data support the use ofVEGF-directed siRNA (also called “anti-VEGF siRNA”) for therapy of ARMD.

Example 7 Generation of an Adeno-Associated Viral Vector for Expressionof siRNA

A “cis-acting” plasmid for generating a recombinant AAV vector fordelivering an siRNA was generated by PCR based subcloning, essentiallyas described in Samulski R et al. (1987), supra. The cis-acting plasmidwas called “pAAVsiRNA.”

The rep and cap genes of psub201 were replaced with the followingsequences in this order: a 19 nt sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and a 19 nt antisense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter. A schematic representation ofpAAVsiRNA is given if FIG. 5.

A recombinant AAV siRNA vector was obtained by transfecting pAAVsiRNAinto human 293 cells previously infected with El-deleted adenovirus, asdescribed in Fisher K J et al. (1996), supra. The AAV rep and capfunctions were provided by a trans-acting plasmid pAAV/Ad as describedin Samulski R et al. (1989), supra. Production lots of the recombinantAAV siRNA vector were titered according to the number of genomecopies/ml, as described in Fisher K J et al. (1996), supra.

Example 8 VEGF-Directed siRNA Inhibits Experimental ChoroidalNeovascularization

The ability of murine VEGF-directed siRNA to inhibit experimentallaser-induced choroidal neovascularization (CNV) in mice was tested asfollows.

The retinas of adult female C57BL/6 mice were laser photocoagulatedusing an 810 nm diode laser (75 um, 140 mw, 0.10 seconds) (OcuLight Six;IRIS Medical, Mountain View, Calif.). Three laser spots were applied toboth eyes of each mouse. Thirty-six hours following laserphotocoagulation, an siRNA targeted to mouse VEGF (“mVEGF1.siRNA”) wasdelivered subretinally or intravitreally to one eye of each mouse. Forsubretinal injection, the siRNA was conjugated with Transit TKOtransfection reagent (Mirus) and mixed with recombinant adenovirus(rAdenovirus). For intravitreal injection, the siRNA was delivered inthe absence of transfection reagent and rAdenovirus. As a control, thecontralateral eyes of each mouse received subretinal or intravitrealinjections of identical formulations with an siRNA targeted to GFP(“GFP1.siRNA”), which has no homology to mouse VEGF.

Fourteen days following laser treatment, all animals were perfused withhigh molecular weight FITC-dextran, choroidal flat mounts were preparedas described above, and the flat mounts were photographed and analyzedmicroscopically in a masked fashion. The area of CNV in each flat mountwas measured with Openlab software (Improvision, Boston, Mass.). Themean areas of CNV in eyes treated with mVEGF1.siRNA were significantlysmaller than those areas from GFP1.siRNA-treated eyes for bothsubretinal (FIG. 6A; P<0.003) and intravitreal (FIG. 6B; P<0.04)delivery.

In a second experiment, the retinas of adult female C57BL/6 mice werelaser photocoagulated as described above, and the animals were dividedinto control and test groups. One day following laser photocoagulation,phosphate buffered saline was delivered intravitreally to the animals ofthe control group, which were perfused with dextran-fluorescein 14 daysafter laser treatment. Choroidal flat mounts were then prepared and theareas of CNV in each flat mount were measured as above.

Fourteen days following laser photocoagulation, mVEGF1.siRNA wasdelivered by intravitreal injection into one eye of each mouse in thetest group. Contralateral eyes were injected with GFP1.siRNA as acontrol. The test group animals were perfused with high molecular weightdextran-fluorescein 21 days after laser treatment. Choroidal flat mountswere then prepared and the areas of CNV in each flat mount weremeasured, as above.

In this latter experiment, the anti-VEGF siRNA was administered duringCNV growth, as opposed to before CNV growth, and thus is morerepresentative of the condition of human patients presenting with wetAMD. As can be seen from FIG. 6, the mean areas of CNV inmVEGF1.siRNA-treated eyes were significantly smaller than those areasmeasured in GFP1.siRNA-treated eyes (FIG. 6C; P<0.05). The mean areas ofCNV in mVEGF1.siRNA-treated eyes at day 21 and control (“PBS”) eyes atday 14 were not significantly different (FIG. 6C; P=0.469).

The results of these experiments indicate that age-related maculardegeneration can be treated with anti-VEGF siRNA.

Example 9 In Vivo RNA Interference of Human VEGF Induced by anti-VEGFsiRNA in Murine RPE Cells

The ability of Cand5 siRNA to induce RNAi of VEGF in vivo over time wasevaluated as follows. AAV.CMV.VEGF, which expresses human VEGF from anadeno-associated viral vector, was generously provided by Dr. A.Auricchio. AAV.CMV.VEGF was injected subretinally and bilaterally ineyes of five C57B1/6 mice. Twenty-eight days after injection ofAAV.CMV.VEGF, Cand5 siRNA was delivered by intravitreal injection intoone eye and control GFP1.siRNA was delivered by intravitreal injectionin the contralateral eye of each animal.

At day 0 (pre-siRNA injection), and at 6, 10 and 14 days after siRNAinjection, the mice were sacrificed and the eyes were snap frozen inliquid nitrogen following enucleation. The eyes were then homogenized inlysis buffer (Roche, Base1, Switzerland), and total protein was measuredusing a Bradford assay, as in Example 5 above. Two mice were used forthe 0 day time point (n=2), and three mice each were used for the 6, 10and 14 day time points (n=3). The samples were normalized for totalprotein prior to assaying for human VEGF by ELISA, according to themanufacturer's recommendations (R&D systems, Minneapolis, Minn.).Percent of VEGF (% VEGF) for each mouse was calculated as theconcentration of VEGF (“[VEGF]”) in the eye injected with Cand5 dividedby the [VEGF] in the eye injected with GFP1.siRNA, multiplied by 100.

As can be seen from FIG. 7, a single injection of Cand5 induced anRNAi-mediated decrease in VEGF levels of approximately 70% by day 6post-siRNA injection, with a reduction in VEGF production ofapproximately 35% continuing through at least day 14 post-siRNAinjection. These results indicate that siRNA directed against human VEGFis capable of inducing RNAi of human VEGF in vivo for a sustained periodof time.

Example 10 In Vivo RNA Interference of VEGF in Monkeys with Anti-VEGFsiRNA

The objectives of this study were to determine the safety and efficacyof Cand5 when administered by single intravitreal injection to malecynomolgus monkeys following induction of CNV. Cand5 was administered inthe vehicle control article to naive male cynomolgus monkeys in thefollowing dose levels: 0 mg/eye (control), 0.07 mg/eye, 0.18 mg/eye,0.35 mg/eye and, and 0.70 mg/eye.

CNV was induced by laser treatment to the maculae of both eyes of eachanimal, and the doses of Cand5 were given shortly following lasertreatment. The animals were evaluated for changes in clinical signs,body weight and ocular condition (extensive ophthalmic examinations,electroretinography and tonometry). Fluorescein angiography wasperformed and blood samples were collected. At the end of the study (Day44), all animals were euthanized and a complete gross necropsy wasperformed. Selected tissues were collected and preserved forhistopathologic evaluation.

No adverse systemic or local (ocular) effects of Cand5 were detectedwhen monkeys were administered a single intravitreal injection into botheyes at doses up to 0.70 mg/eye following laser lesioning of the maculaand during subsequent development of CNV.

Example 11 In Vitro RNA Interference of VEGF with Anti-VEGF siRNA inHuman Embryonic Kidney 293 Cells

Human embryonic kidney 293 cells (obtained from ATCC, Manassas, Va.)were cultured in Dulbecco's Modified Eagle Medium (DMEM; obtained fromCellgro, Herndon, Va.) with 10% fetal bovine serum (FBS; from JRHBiosciences, Lenexa, Kans.) and an antibiotic-antimycotic reagent, usedfor the prevention of cell culture growth contaminants (from Gibco,Carlsbad, Calif.).

siRNAs were synthesized by Integrated DNA Technologies (Coralville,Iowa). The siRNA target sequences are shown in Table 2. An additionalsiRNA was used in this study that targets the gene of enhanced greenfluorescent protein (EGFP) as a negative control.

TABLE 2 GC Nucleotide Name Content Start Site Target Sequence 5′ -3′hVEGF#1 58% 92 aaggaggagggcagaatcatc (SEQ ID NO: 81) hVEGF#2 42% 124aagttcatggatgtctatcag (SEQ ID NO: 47) hVEGF#3 58% 162aatcgagaccctggtggacat (SEQ ID NO: 48) hVEGF#4 42% 301aacatcaccatgcagattatg (SEQ ID NO: 50) hVEGF#5 58% 338aaggccagcacataggagaga (SEQ ID NO: 52) hVEGF#6 42% 380aatgtgaatgcagaccaaaga (SEQ ID NO: 82) hVEGF#7 37% 396aaagaaagatagagcaagaca (SEQ ID NO: 56) hVEGF#8 32% 450aaagcatttgtttgtacaaga (SEQ ID NO: 83) hVEGF#9 42% 467aagatccgcagacgtgtaaat (SEQ ID NO: 84) hVEGF#10 53% 498aaacacacactcgcgttgcaa (SEQ ID NO: 85) Cand5 63% 328aaacctcaccaaggccagcac (SEQ ID NO: 51)

siRNA Transfection and Hypoxia Induction In Vitro. Human 293 cells werecultured in 24 well plates at 37° C. with 5% CO₂ overnight. The nextday, transfections were performed when cells were about 50%-70%confluent. Cells were transfected with siRNAs directed against humanVEGF. siRNAs were mixed in a CaPi reagent and added to 20 μl of 250 mMCaCl₂ solution. The siRNA/CaCl₂ mixture was added drop-wise to 20 μl of2× Hanks Balanced Salt Solution (HBS), while mixing by vortex. ThesiRNA/CaCl₂/HBS complex was added directly to the medium in each well(300 μL/well). After a 4-hour incubation at 37° C., the medium wasremoved, and the cells were further incubated with 10% DMSO-containingserum-free medium (300 μL/well at room temperature for 1-2 minutes).This medium was then removed, and the cells were fed again with growthmedium (500 μL/well). Negative controls included transfection reagentlacking siRNA and nonspecific siRNA (EGFP1 siRNA). For screeningexperiments siRNAs were used at a concentration of 25 nM. For doseresponse experiments, siRNAs were used at concentrations of 1 nM, 5 nMand 25 nM. Hypoxia was induced with desferrioxamine at a finalconcentration of 130 uM 4 hours after transfection was performed.Desferrioxamine mimics a hypoxic state, as it is proposed to disruptnormal oxygen-sensing pathways in mammalian cells by inhibitingheme-Fe2+ interactions.

VEGF Protein Quantification. Approximately 48 hours post transfection,the supernatant was removed from all wells and a human VEGF ELISA (R & Dsystems, Minneapolis, Minn.) was performed on the 293 cells as describedin the Quantikine human VEGF ELISA protocol. VEGF-specific antibody wasadded to each well causing color development in proportion to the amountof VEGF bound to the plate. ELISA results were read on an AD340 platereader at 450 nm (Beckman Coulter).

Results. Human VEGF siRNAs Suppresses Hypoxia-Induced Up-regulation ofHuman VEGF Protein in 293 Cells. Human VEGF was upregulated by thedesferrioxamine-mediated induction of hypoxia. Readings of OD 450 nmreflected the human VEGF protein levels in cell samples. Thehypoxia-induced increase of hVEGF protein levels were significantlyreduced in cells transfected with all of the human VEGF siRNAs (FIG. 8).No effect on hVEGF levels were observed with transfections withnonspecific siRNA (EGFP siRNA) or mock transfections without siRNA. Doseresponse studies were performed on Cand5, hVEGF#1, hVEGF#2, hVEGF#3,hVEGF#4, hVEGF#6 and hVEGF#7 (FIG. 9).

Example 12 In Vitro RNA Interference of VEGF Isoforms

VEGF_(165b) has been identified as an endogenous anti-angiogenic VEGFisoform. siRNA were designed to selectively inhibit certain VEGFisoforms, such as VEGF₁₆₅, but spare VEGF_(165b).

Methods: ARPE19 cells were seeded in 24 well plates (50,000 cells perwell). Eighteen to twenty-four hours post-seeding, cells were 50-75%confluent and used for transfection. Fourteen human VEGF-A specificsiRNAs were designed and tested. Cells were transfected with the siRNAs(25 nM) using RiboJuice™ siRNA Transfection Reagent (Novagen) followingthe manufacturer's protocol. Specifically, for a single well of cells,40.5 μL serum free OPTI-MEM® was pipetted into an eppendorf tube then 2μL of RiboJuice™ was added to the OPTI-MEM. The solution was mixed bygentle vortexing and centrifuged briefly to collect contents at bottomof the tube and incubated at room temperature for 5 minutes. siRNA (7.5μL of a 1 μM stock) was added to the RiboJuice™/medium mix and gentlymixed and briefly centrifuged to collect contents at the bottom of thetube. The mixture was incubated at room temperature for 15 minutes.During the incubation, media was removed from cells and replaced with250 μL of fresh complete ARPE19 growth media (DMEM/F12; 10% FBS, 1%penicillin/streptomycin). After the 15 minute incubation thesiRNA/RiboJuice™/medium mixture (50 μL) was added dropwise to the cells.The final concentration of siRNA in the 300 μL volume was 25 nM. Cellswere maintained at 37° C., 5% CO₂ for 24 hours. In additionalexperiments, reactions were scaled up to transfect cells in triplicatewells with each siRNA. 24 hours post-transfection, the transfectionmixture was removed and the cells were treated with 500 μLs of serumfree DMEM/F12, DMEM/F12 containing 10 ng/mL human recombinant TGFβII orDMEM/F12 containing 10 ng/mL TGFβII and 5 μg/mL cycloheximide. The cellswere returned to 37° C. and 5% CO₂ for an additional 24 hours.Afterwards, the media was removed from the cells and analyzed forprotein expression by ELISA (Quantikine human VEGF ELISA kit (R&DSystems)). Media was removed from cells and collected in eppendorf tubesand placed on ice and immediately analyzed for VEGF protein via ELISA,or stored at −80° C. and analyzed for VEGF protein at a later timepoint.

Based on these results, a select number of siRNA candidates were putthrough an additional transfection screen. Cells were collected, RNAextracted, and semi-quantitative RT-PCR was performed to determine thesiRNAs' inhibitory effect on VEGF₁₆₅, VEGF_(165b), VEGF₁₂₁ and VEGF₁₈₉.GAPDH housekeeping gene expression was used as a control. Specifically,after removing the media from the wells, 200 μLs of lysis/bindingsolution from the RNAqueous Kit (Ambion)was added to each well. RNA wasquantified via spectrophotometry (OD 260 nM). The lysed cells werecollected and RNA was extracted following the manufacturer's protocol.RNA was reverse transcribed using SuperScript™ III Reverse Transcriptase(Invitrogen) according to the manufacturer's protocol. cDNA was analyzedfor GAPDH, VEGF₁₆₅, VEGF_(165b), VEGF₁₂₁ and VEGF₁₈₉ using PCR. Primersused for PCR are shown in Table 3.

TABLE 3 Primer Name Description Sequence 5′-3′ P121Reverse primer VEGF121 GGCTTGTCACATTTTTCTTG (SEQ ID NO: 131) P165Reverse primer VEGF165 CCCACAGGGATTTTCTTGTC (SEQ ID NO: 132) P189Reverse primer VEGF189 CTTTCCCTTTCCTCGAACTG (SEQ ID NO: 133) hVEGF-EForward primer used for GCTACTGCCATCCAATCGAG (SEQ ID NO: 134)VEGF121, VEGF165 & VEGF 189 P165bR Reverse primer forGTCTTTCCTGGTGAGAGATC (SEQ ID NO: 135) VEGF165b hVEGF-AForward primer for CTGTCTTGGGTGCATTGGAG (SEQ ID NO: 136) VEGF165bGAPDH-B Reverse primer GAPDH GAGGCAGGGATGATGTTCTG (SEQ ID NO: 137)GAPDH-A Forward primer GAPDH CATGGCAAATTCCATGGCAC (SEQ ID NO: 138)

For PCR analysis, 3 μL cDNA was combined with 1 μL of each appropriateforward (10 μM) and reverse primer (10 μM) primer and 45 μL of PlatinumPCR Supermix (Invitrogen) such that the final concentration of eachprimer was 200 nM. The cDNA was amplified in a thermocycler with thefollowing PCR conditions:

Step 1: 94° C. for 2 minutes

Step 2: 94° C. for 15 seconds

Step 3: 55° C. for 30 seconds

Step 4: 72° C. for 30 seconds

Step 5: Repeat steps 2-4 30 times for GAPDH, VEGF₁₆₅, VEGF₁₂₁ andVEGF₁₈₉ or 35 times for VEGF_(165b)

Step 6: 72° C. for 10 minutes

Step 7: 4° C.

PCR product was then visualized on a 2% agarose gel prepared in 1×TAEbuffer.

Results: Treatment of ARPE19 cells with TGFβII induced VEGF productionin ARPE19 cells and ELISA results demonstrated several siRNA candidatesinhibited the production of TGFβII-induced VEGF in ARPE19 cells. RT-PCRconfirmed that 2 candidates inhibited production of VEGF₁₆₅, VEGF₁₂₁ andVEGF₁₈₉, but spared VEGF_(165b). As shown in FIG. 12 (pg/mL hVEGF) and13 (% knockdown hVEGF), VEGF siRNA candidates (Table 2) were screenedfor the ability to inhibit VEGF protein production by ARPE19 cells astested by ELISA. Cells were treated with 10 ng/mL TGFβII to upregulateVEGF production. ELISA measured total VEGF protein and was not selectivefor any particular splice variant. Several candidates (OPK-HVB-004,OPK-HVB-010, and OPK-HVB-011) demonstrate an inhibitory effect andwarranted further study. As shown in FIG. 14 (pg/mL hVEGF) and 15 (%knockdown hVEGF), a secondary screen of VEGF production using the samemethods as in FIGS. 12 and 13 demonstrated that OPK-HVB-004 andOPK-HVB-010 inhibited VEGF protein production and warranted furtherinvestigation.

FIGS. 16, 24 and 27 demonstrate a dose response efficacy of human VEGFknockdown with several candidates (OPK-HVB-004, OPK-HVB-010, andOPK-HVB-012) at varying concentrations.

FIG. 17 demonstrates downregulation of human VEGF over one week (7 days)of several candidates (OPK-HVB-004, OPK-HVB-010, and OPK-HVB-012).

As a control, GAPDH RT-PCR was performed on variously treated cells asshown in FIG. 18. Although the actual amount of RNA present was notquantified, the procedures are semi-quantitative when compared to thereference control lane 3. Specifically, downregulation of RNA productionis demonstrated when a band appears fainter. In this experiment, samplesin Lanes 2-11 were treated with 10 ng/mL TGFβII to upregulate theproduction of VEGF. The FAM-GAPDH siRNA downregulated GAPDH message(lane 4), while the other treatments have no effect on GAPDH mRNA, thusconfirming that there is no variability in total RNA production in thetreated cells.

VEGF₁₆₅ isoform RT-PCR was also performed on the treated cells as shownin FIG. 19. Samples in Lanes 2-11 were treated with 10 ng/mL TGFβII toupregulate the production of VEGF. 25 nM bevasiranib (lane 6), which isknown to downregulate all VEGF isoforms, 25 nM OPK-HVB-004 (lane 7) and25 nM OPK-HVB-010 (lane 8), downregulated the production of VEGF₁₆₅ mRNAfollowing induction with TGFβII (lane 2), as demonstrated by the bandsbeing lighter than control in lane 3.

VEGF₁₈₉ isoform RT-PCR was also performed as shown in FIG. 20. Samplesin Lanes 2-11 were treated with 10 ng/mL TGFβII to upregulate theproduction of VEGF.

25 nM bevasiranib (lane 6), 25 nM OPK-HVB-004 (lane 7) and 25 nMOPK-HVB-010 (lane 8) downregulated the production of VEGF₁₈₉ mRNAfollowing induction with TGFβII (lane 2) as demonstrated by the bandsbeing lighter than control in lane 3.

VEGF₁₂₁ isoform RT-PCR was then performed as shown in FIG. 21. Samplesin Lanes 2-11 were treated with 10 ng/mL TGFβII to upregulate theproduction of VEGF.

VEGF₁₂₁ mRNA was downregulated in lane 6 (25 nM bevasiranib) asdemonstrated by the bands being lighter than control in lane 3.

Finally, VEGF_(165b) isoform RTPCR was performed as shown in FIG. 22.

Samples in Lanes 2-11 were treated with 10 ng/mL TGFβII to upregulatethe production of VEGF. As an initial matter, the double banding >600 bpwas determined to be artifactual. However, VEGF_(165b) mRNA isdownregulated by bevasiranib (lane 6) as shown by the bands beingfainter that the control of lane 3. In contrast, bands for OPK-HVB-004(lane 7) and OPK-HVB-010 (lane 8) were not fainter that control in lane3. Thus, these siRNA constructs preserved VEGF_(165b) expression whilealso being able to inhibit various other VEGF isoforms. Thus, siRNAssparing VEGFA_(165b) can be synthesized and may be more efficacious thensiRNAs that knockdown all VEGF-A isoforms. VEGF_(165b) sparing siRNAsmay be potent therapeutic candidates for the treatment of ocularneovascularization.

Example 13 Cytokine Profile Following Treatment with siRNAs

The cytokine secretion profile of ARPE19 cells following treatment withpolyinosinic-polycytidylic acid sodium salt [Poly (LC)], a dsRNAanalogue was determined Further tests to determined whether or notsiRNAs behaved like Poly (I:C) and caused the cells to produce the samecytokines were conducted.

Methods. ARPE19 cells were seeded in 24 well plates (50,000 cells perwell). Twenty-four hours later, media was removed and cells were treatedwith Poly (I:C); 0-1000 mg/mL (Sigma, St. Louis, Mo.) or polydeoxyinosinic-deoxycytidylic acid sodium salt [Poly (dI:dC); 50mU/mL-800mU/mL] (Sigma), prepared in serum free DMEM/F12(1:1) (Invitrogen,Carlsbad, Calif.). Forty-eight hours post-treatment, media was collectedfrom cells and analyzed for IFN-α, IFN-β, IFN-γ, IL-8, IL-6, TNFα, ICAM,IL-12 and MCP-1 via ELISA (QUANTIKINE® Immunoassays for IFN-7, IL-8,IL-6, TNFα, ICAM, IL-12 and MCP-1, R&D Systems, Minneapolis, Minn.);VeriKine™ ELISA kits for IFN-α and IFN-(3, PBL Biomedical Laboratories,Piscataway, N.J.) according to the manufacturers' protocols.

ARPE19 cells were transfected with bevasiranib, OPK-HVB-004,OPK-HVB-009, OPK-HVB-010 and OPK-HVB-012 (Dhamacon/Thermo Scientific,Chicago, Ill.). Cells were seeded in 24 well plates (40,000 cells perwell). 24 hours later, cells were transfected with 25 nM siRNA usingRiboJuice™ Transfection Reagent (Novagen/EMD, San Diego, Calif.)according to the manufacturer's protocol. 24 hours post-ransfection,cells were treated with 10 ng/mL human recombinant TGFβII (R&D Systems).48 hours post-transfection (ie. 24 hours post-TGFβII treatment), mediawas collected and cytokine levels were analyzed, as described above.Additionally, media was analyzed for hVEGF via ELISA (R&D Systems).Results are shown in FIG. 23.

Conclusions. Based upon the foregoing it is suggested that (ii) ARPE19cells produce several inflammatory cytokines in response to Poly (I:C),a dsRNA analogue, but do not produce three key mediators, IFN-α, IFN-13or IFN-7; (ii) ARPE19 cells can be used to study the inflammatorypotential and specific effects of dsRNAs such as siRNAs; and (iii)OPK-HVB-009 an OPK-HVB-010 did not cause ARPE19 cells to secrete any ofthe cytokines tested, suggesting they may have a low inflammatorypotential.

Example 14 Dose Response Curves Shows Specificity of siRNAs

A dose response curve was generated using various siRNAs, 21-mers, asshown in FIGS. 26 and 27. A dose response was seen with certain siRNASindicating a specific response to the siRNAs used. A dose response curvewas also generated for OPK-HVB-009 as shown in FIGS. 25 and 26. Thecells were treated and transfected as described in Examples 12 and 13.Cells were seeded in 24 well plates (40,000 cells/well). Additionally,different concentrations were used, and therefore, the volumes ofOPTI-MEM, RiboJuice™, and siRNA were adjusted accordingly when preparingthe 50 μl transfection mix.

Example 15 Stability of siRNAs

ARPE19 cells were transfected with siRNAs that had been stored undervarious conditions as shown in FIGS. 28, 29, 30 31, and 32. The cellswere transfected as described in Examples 12 and 13. It was found thatthe siRNA molecules were stable under various conditions as shown inFIGS. 28, 29, 30, 31, and 32. For example, 7.5 μM siRNA was aliquotedinto 3 tubes and each tube was stored at a different temperature (37°C., room temperature, 4° C.) for up to 8 weeks. Aliquots of each tubewere collected at predetermined time points (24 hrs, 48 hrs and thenweekly). Upon collection aliquots were stored at −80° C. Each aliquotwas subsequently tested for efficacy in ARPE19 cells to see if thesiRNAs maintained their stability under the different environmentalconditions. siRNAs were transfected into ARPE19 cells using the methodsdescribed in Example 12 where 40,000 cells were seeded per well.

Example 16 Cross-Species Down Regulation of VEGF

C6 cells were seeded in 24 well plates (P12, 40,000 cells per well).Eighteen to twenty-four hours post-seeding, cells were 50-70% confluentand used for transfection. Cells were transfected with OPK-HVB-004,OPK-HVB-009, OPK-HVB-010 and OPK-HVB-012 using the RiboJuice™ siRNATransfection Reagent (Novagen) following the manufacturer's protocol.Briefly, for a single well serum-free OPTI-MEM (40.5 μL-47 μL) waspipetted into an eppendorf tube and then 2 μL of RiboJuice™ were addedto the OPTI-MEM (Gibco). The solution was mixed by gentle vortexing andcentrifuged briefly to collect the contents at the bottom of the tubeand incubated at room temperature for 5 min siRNA (0.3 μL-7.5 μL of a100 nM or 1 μM stock) was added to the RiboJuice™/medium mix and gentlymixed and briefly centrifuged to collect contents at the bottom of thetube. The mixture was incubated at room temperature for 15 minutes.During the incubation, media was removed from cells and replaced with250 μL of fresh C6 growth media (F-12 Kaighn's, 2.5% fetal calf serum;15% horse serum, 1% penicillin/streptomycin). After the 15 minincubation, the siRNA/RiboJuice™/medium mixture (50 μL) was addeddropwise to the cells. The plate was gently rocked to ensure thecomplexes were evenly dispersed throughout the well. The finalconcentration of siRNA in the 300 μL volume was 250 pM, 500 ppM, 1 nM, 5nM or 25 nM. Cells were maintained at 37° C., 5% CO2 for 24 hours. Allvolumes were scaled up such that each siRNA was tested at eachconcentration in triplicate. 24 hours post-transfection, thetransfection mixture was removed and cells were treated with 500 μLs offresh C6 growth media or with fresh C6 growth media supplemented with 10ng/mL human recombinant TGFβII. The cells were returned to 37° C., 5%CO₂ for an additional 24 hours. Afterwards the media was removed fromthe cells and analyzed for protein expression by ELISA (Quantikine ratVEGF ELISA kit, R&D Systems).

NIH3T3 cells were seeded in 24 well plates (P2-P6, 40,000 cells perwell). Eighteen to twenty-four hours post-seeding, cells were 50-70%confluent and used for transfection. Cells were transfected with siRNAsusing Lipofectamine™ Reagent 2000 (Invitrogen) following themanufacturer's protocol. Briefly for a single well, siRNA (1 μM or 7.5μM) was diluted in 50 μL OPTI-MEM in an eppendorf tube and gently mixedand vortexed. In a second eppendorf tube 1 μL of Lipofectamine 2000 wascombined with 49 μL of OPTI-MEM. The mixture was gently mixed andvortexed and incubated for 5 minutes at room temperature. After the 5minutes, the diluted siRNA (50 μL volume) was added to the dilutedLipofectamine 2000 (50 μL). The contents were mixed gently and incubatedat room temperature for 20 minutes. During the 20 minute incubation,media was removed from the cells and replaced with 500 μLs of freshNIH3T3 growth media (DMEM, 10% fetal calf serum). After the 20 minutesthe siRNA-Lipofectamine 2000 complex (100 μL) was added dropwise to thecells. The plate was gently rocked to ensure the complexes were evenlydispersed throughout the well. The cells were then incubated at 37° C.,5% CO₂ for 24 hours. The final concentration of siRNA in the 500 μLvolume was 1 nM, 5 nM or 25 nM. 24 hours post-transfection, thetransfection mixture was removed and cells were treated with 500 μLs offresh DMEM or with fresh DMEM supplemented with 10 ng/mL humanrecombinant TGFβII. The cells were returned to 37° C., 5% CO₂ for anadditional 24 hours. Afterwards the media was removed from the cells andanalyzed for protein expression by ELISA (Quantikine mouse VEGF ELISAkit, R&D Systems).

Results of the experiments are shown in FIGS. 34, 35 and 39. OPK-HVB-004and OPK-HVB-009 were able to inhibit VEGF secretion by C6 cells as shownin FIG. 34. Similar experiments were done in mouse cells (NIH3T3) andOPK-HVB-004, OPK-HVB-009, and OPK-HVB-010 were able to inhibit secretionof mouse VEGF as shown in FIGS. 35 and 39.

Example 17 Comparison of Different siRNAs

21mer siRNAs comprising an overhang were compared to a 19mer blunt-endcounterpart. ARPE19 cells were transfected with the different siRNAs asdescribed in Examples 12, 13, and 14 and VEGF production was measured.The Blunt end counterpart was found to knockdown VEGF production inARPE19 cells equally effective as the 21mer as shown in FIG. 36. Forexample, a blunt end version of bevasiranib comprising SEQ ID NO: 119and SEQ ID NO: 120 was equally effective at knocking down VEGFproduction as shown in FIG. 36.

Example 18 Screen of 19mers Comprising 17 bp and an Overhang can InhibitVEGF Production

siRNAS comprising a 17mer and a dTdT overhang were transfected in ARPE19cells as described in Examples 12, 13, and 14. Several siRNAs were foundto inhibit VEGF production as shown in FIG. 37.

Example 19 Dose Response of siRNAs

19mers comprising a blunt end or an overhang 19mer (17 bp+dTdT over)were transfected into ARPE19 cells at various doses as shown in FIG. 38.A dose response curve was generated by measuring VEGF secretion asdescribed in Examples 12, 13 and 16. The dose response seen indicatesthat the response to the siRNAs is specific to the siRNA and notgenerated by a non-specific siRNA response. The results can be seen inFIG. 38. Blunt end siRNAs tested in NIH3T3 cells showed a specific doseresponse. (See FIG. 39).

Example 20 Effect of siRNAs on VEGF mRNA Expression

ARPE19 cells were transfected with siRNAs (final concentration siRNA=25nM). Cells were treated with 10 ng/mL TGFβII to upregulate production ofhVEGF. RNA was isolated from cells and reverse transcription PCR wasperformed to amplify GAPDH (FIG. 40A; 472 bp fragment), VEGF₁₆₅ (FIG.40B; 284 bp fragment) and VEGF_(165b) (FIG. 40C; 199 bp fragment). TheCy3-GAPDH siRNA (FIG. 40A, Lane 4) silenced GAPDH message whereas theother treatments had no effect. OPK-HVB-004, OPK-HVB-009 and OPK-HVB-010(FIG. 40B, Lanes 6, 7 and 8) and OPK-HVB-004be, OPK-HVB-009be andOPK-HVB-010be (FIG. 40B, Lanes 10, 11 and 12) silenced VEGF₁₆₅ message.Bevasiranib (FIG. 40C, Lane 4) silenced VEGF_(165b) whereas OPK-HVB-004,OPK-HVB-009, OPK-HVB-004be and OPK-HVB-009be (FIG. 13C, Lanes 5, 6, 8and 9) preserved levels of VEGF_(165b) (See FIG. 40).

Example 21 Efficacy of siRNAs in Rat C6 Cells

Rat C6 cells were transfected with siRNAs (final concentration siRNA=25nM). Cells were treated with 10 ng/mL TGFβII to upregulate production ofrat VEGF. Levels of total secreted VEGF were measured in media viaELISA. Percent knockdown reflects the level of VEGF produced by thecells relative to cells treated with RiboJuice™. OPK-HVB-004 andOPK-HVB-004be were the most effective in reducing levels of rat VEGF.

1. An isolated siRNA comprising a sense RNA strand and an antisense RNAstrand, wherein the sense and the antisense RNA strands form an RNAduplex, and wherein the sense RNA strand comprises a nucleotide sequenceidentical to a target sequence of about 19 to about 25 contiguousnucleotides in human VEGF mRNA, wherein the sense RNA strand comprises anucleotide sequence that consists of SEQ ID NO: 119, and the antisensestrand comprises a nucleotide sequence that consists of SEQ ID NO: 120.2. The siRNA of claim 1, wherein the first and second RNA strandsforming the RNA duplex are covalently linked by a single-strandedhairpin.
 3. The siRNA of claim 1, wherein the siRNA further comprisesnon-nucleotide material.
 4. The siRNA of claim 1, wherein the first andsecond RNA strands are stabilized against nuclease degradation.
 5. Apharmaceutical composition comprising an effective amount of an isolatedsiRNA comprising a sense RNA strand and an antisense RNA strand, whereinthe sense and the antisense RNA strands form an RNA duplex, and whereinthe sense RNA strand comprises a nucleotide sequence that consists ofSEQ ID NO: 119, and the antisense strand comprises a nucleotide sequencethat consists of SEQ ID NO:
 120. 6. The pharmaceutical composition ofclaim 5, wherein the first and second RNA strands are stabilized againstnuclease degradation.
 7. A method of treating an angiogenic disease in asubject comprising: administering to a subject an effective amount of ashort interfering ribonucleic acid (siRNA) comprising a sense RNA strandand an antisense RNA strand, wherein the sense and the antisense RNAstrands form an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence identical to a target sequence of about 19 to about25 contiguous nucleotides in human vascular endothelial growth factor(VEGF) mRNA, and wherein the sense RNA strand comprises a nucleotidesequence that consists of SEQ ID NO: 119, and the antisense strandcomprises a nucleotide sequence that consists of SEQ ID NO: 120, suchthat angiogenesis associated with the angiogenic disease is inhibited.8. The method of claim 7, wherein the angiogenic disease comprises atumor associated with a cancer.
 9. The method of claim 8, wherein thecancer is selected from the group consisting of breast cancer, lungcancer, head and neck cancer, brain cancer, abdominal cancer, coloncancer, colorectal cancer, esophagus cancer, gastrointestinal cancer,glioma, liver cancer, tongue cancer, neuroblastoma, osteosarcoma,ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma,Wilm's tumor, multiple myeloma, skin cancer, lymphoma, and blood cancer.10. The method of claim 7, wherein the angiogenic disease is selectedfrom the group consisting of diabetic retinopathy, age-related maculardegeneration, and inflammatory diseases.
 11. The method of claim 10,wherein the inflammatory disease is psoriasis or rheumatoid arthritis.12. The method of claim 7, wherein the angiogenic disease is age-relatedmacular degeneration.
 13. The method of claim 7, wherein thepharmaceutical composition is administered in combination with apharmaceutical agent for treating the angiogenic disease, whichpharmaceutical agent is different from the short interfering ribonucleicacid (siRNA).
 14. The method of claim 7, wherein the angiogenic diseaseis cancer, and the pharmaceutical agent comprises a chemotherapeuticagent.
 15. The method of claim 14, wherein the chemotherapeutic agent isselected from the group consisting of cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin, andtamoxifen.
 16. The method of claim 7, wherein the pharmaceuticalcomposition is administered to a subject in combination with anothertherapeutic method designed to treat the angiogenic disease.
 17. Themethod of claim 7, wherein the angiogenic disease is cancer, and thepharmaceutical composition is administered in combination with radiationtherapy, chemotherapy or surgery.
 18. A method of inhibiting expressionof human vascular endothelial growth factor (VEGF) comprising:administering to a subject an effective amount of a short interferingribonucleic acid (siRNA) comprising a sense RNA strand and an antisenseRNA strand, wherein the sense and the antisense RNA strands form an RNAduplex, and wherein the sense RNA strand comprises a nucleotide sequenceidentical to a target sequence of about 19 to about 25 contiguousnucleotides in human vascular endothelial growth factor (VEGF) mRNA andwherein the sense RNA strand comprises a nucleotide sequence thatconsists of SEQ ID NO: 119, and the antisense strand comprises anucleotide sequence that consists of SEQ ID NO:
 120. 19. The method ofclaim 18, wherein the effective amount comprises from about 1 nM toabout 100 nM of the short interfering ribonucleic acid (siRNA).
 20. Themethod of claim 18, wherein the pharmaceutical composition furthercomprises a delivery reagent.
 21. The method of claim 18, wherein thedelivery agent is selected from the group consisting of lipofectin,lipofectamine, cellfectin, polycations, and liposomes.
 22. The method ofclaim 21, wherein the delivery agent is a liposome.
 23. The method ofclaim 22, wherein the liposome comprises a ligand which targets theliposome to cells at or near the site of angiogenesis.
 24. The method ofclaim 23, wherein the ligand binds to receptors on tumor cells orvascular endothelial cells.
 25. The method of claim 23, wherein theligand comprises a monoclonal antibody.
 26. The method of claim 22,wherein the liposome is modified with an opsonization-inhibition moiety.27. The method of claim 26, wherein the opsonization-inhibiting moietycomprises a PEG, PPG, or derivatives thereof.
 28. The method of claim18, wherein the short interfering ribonucleic acid (siRNA) is expressedfrom a recombinant plasmid.
 29. The method of claim 18, wherein theshort interfering ribonucleic acid (siRNA) is expressed from arecombinant viral vector.
 30. The method of claim 29, wherein therecombinant viral vector comprises an adenoviral vector, anadeno-associated viral vector, a lentiviral vector, a retroviral vector,or a herpes virus vector.
 31. The method of claim 30, wherein therecombinant viral vector is pseudotyped with surface proteins fromvesicular stomatitis virus, rabies virus, Ebola virus, or Mokola virus.32. The method of claim 29, wherein the recombinant viral vectorcomprises an adeno-associated viral vector.
 33. The method of claim 18,wherein the pharmaceutical composition is administered by an enteraladministration route.
 34. The method of claim 33, wherein the enteraladministration route is selected from the group consisting of oral,rectal, and intranasal.
 35. The method of claim 18, wherein thepharmaceutical composition is administered by a parenteraladministration route.
 36. The method of claim 35, wherein the parenteraladministration route is selected from the group consisting ofintravascular administration, peri- and intra-tissue injection,subcutaneous injection or deposition, subcutaneous infusion, and directapplication at or near the site of neovascularization.
 37. The method ofclaim 35, wherein the intravascular administration is selected from thegroup consisting of intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature.
 38. A method of degrading humanvascular endothelial growth factor (VEGF) mRNA comprising: administeringto a subject an effective amount of a short interfering ribonucleic acid(siRNA) comprising a sense RNA strand and an antisense RNA strand,wherein the sense and the antisense RNA strands form an RNA duplex, andwherein the sense RNA strand comprises a nucleotide sequence identicalto a target sequence of about 19 to about 25 contiguous nucleotides inhuman vascular endothelial growth factor (VEGF) mRNA and wherein thesense RNA strand comprises a nucleotide sequence that consists of SEQ IDNO: 119, and the antisense strand comprises a nucleotide sequence thatconsists of SEQ ID NO: 120.